Therapeutic compounds

ABSTRACT

The invention relates to protein binding interacting/binding compounds and methods of identifying and using them. The invention further relates to pharmaceutical compositions and methods for treating 5-HT2C disorders, including diseases and disorders mediated by GPCRs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/934,743, filed Jun. 15, 2007, and 61/070,386, filedMar. 21, 2008. The contents of these applications are incorporatedherein by reference in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported in part by United States Public Health ServiceGrant MH068655. The government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Serotonin (5-hydroxytryptamine, 5HT) mediates a wide variety of centraland peripheral psychological and physiological effects through 14mammalian 5HT receptor subtypes that are grouped into the 5HT₁-5HT₇families (Sanders-Bush and Mayer, 2006). The 5HT₂ family consists of the5HT_(2A), 5HT_(2B), and 5HT_(2C) membrane-bound G protein-coupledreceptors (GPCRs) that signal primarily through Gα_(q) to activatephospholipase (PL) C and formation of inositol phosphates (IP) anddiacylglycerol (DAG) second messengers (Raymond et al., 2001). The human5HT_(2C) receptor (Saltzman et al., 1991) apparently is foundexclusively in brain where it is widely expressed and putativelyinvolved in several (patho)-physiological and psychological processes,including, ingestive behavior (Tecott et al., 1995), cocaine addiction(Fletcher et al., 2002; Rocha et al., 2002; Muller and Huston, 2006),sleep homeostasis (Frank et al., 2002), anxiety (Kennett et al., 1994;Sard et al., 2005; Heisler et al., 2007), depression (Tohda et al.,1989; Palvimaki et al., 1996), epilepsy (Heisler et al., 1998),Alzheimer's disease (Arjona et al., 2002; Stein et al., 2004), motorfunction (Heisler and Tecott, 2000; Segman et al., 2000), psychosis(Marquis et al., 2007; Siuciak et al., 2007) and response toantipsychotic drugs (Veenstra-VanderWeele et al., 2000; Reynolds et al.,2005). Thus, the importance of the 5HT_(2C) receptor as apharmacotherapeutic target has been apparent for about 10 years,however, no 5HT_(2C)-specific drugs have been developed.

One challenge regarding drug discovery targeting the 5HT_(2C) receptoris that this GPCR shares a transmembrane domain (TMD) sequence identityof about 80% with the 5HT_(2A) receptor and about 70% with the 5HT_(2B)receptor (Julius et al., 1988; 1990). The highly conserved TMDs andsimilar second messenger coupling has made development of agonistligands selective for the 5HT_(2C) receptor especially difficult.Nevertheless, there is compelling evidence that activation of 5HT_(2C)receptors reduces food intake and leads to anti-obesity effects. Forexample, 5-HT_(2C) knockout mice demonstrate increased feeding andobesity, and, they are resistant to the anorectic effects ofd-fenfluramine (Tecott et al., 1995; Vickers et al., 1999; 2001; Heisleret al., 2002). Fenfluramine now is banned, because, although peopleusing the drug showed weight loss due to activation of brain 5HT_(2C)receptors, fenfluramine also activates 5HT_(2A) receptors that may leadto adverse psychiatric (hallucinogenic) effects (Nichols, 2004) and5HT_(2B) receptors which causes valvular heart disease (Connolly et al.,1997; Fitzgerald et al., 2000; Rothman et al., 2000; Roth, 2007) andpulmonary hypertension (Pouwels et al., 1990; Launay et al.,2002)—fatalities have resulted from the 5HT_(2B)-mediated effects.

Although an agonist ligand truly selective for 5HT_(2C) vs. 5HT_(2A)and/or 5HT_(2B) receptors has not been reported until this paper, it hasbeen possible to partially elucidate the role of brain 5HT_(2C)receptors to attenuate cocaine use and dependence using very selective(i.e., at least 100-fold) 5HT_(2A) and 5HT_(2C) antagonists in ratcocaine self-administration paradigms. For example, the selective5HT_(2A) antagonist M100907 (Kehne et al., 1996) does not alterresponding rate for cocaine self-administration but the selective5HT_(2C) antagonist SB242084 (Bromidge et al., 1997) increases the rateof cocaine self-administration dose-dependently (Fletcher et al., 2002).The tremendous potential of 5HT_(2C) agonist pharmacotherapy forpsychostimulant addiction now is widely recognized (Bubar andCunningham, 2006).

The pharmacotherapeutic relevance of the 5HT_(2C) receptor in obesityand neuropsychiatric disorders such as psychostimulant addiction hasstimulated intense interest by pharmaceutical companies to develop aselective 5HT_(2C) agonist, however, all 5HT_(2C) agonists reported sofar also activate 5HT_(2A) and/or 5HT_(2B) receptors (Nilsson, 2006).Nevertheless, the 5HT₂ agonist lorcaserin (APD356) recently went toPhase III clinical trials for obesity treatment even though it has onlya modest 15-fold selectivity for activation of 5HT_(2C) receptors over5HT_(2A) receptors (Jensen, 2006; Smith et al., 2006). Results reportedhere, however, document that a novel compound synthesized in ourlaboratories,(1R,3S)-(−)-trans-1-phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene(PAT; FIG. 1), is a full efficacy agonist at human 5HT_(2C) receptors,plus, it is an antagonist at 5HT_(2A) and 5HT_(2B) receptors.

G Protein-Coupled Receptors (GPCRs) can activate more than more type ofG protein that results in multiple physiological/pharmacologicaleffects, both pharmacotherapeutic and untoward side effects (Moniri etal., Journal of Pharmacology and Experimental Therapeutics, 311:274-281(2004)). The phenomenon of multiple signaling pathways associated with asingle GPCR can be described within the framework of the three-statemodel of GPCR activation, wherein, GPCRs isomerize between inactive andconstitutively active states. GPCR activation causes dissociation ofheterotrimeric (α,β,γ) G protein subunits—the Gα subunit can thenactivate transducer protein (e.g., PLC, AC) to alter second messengerconcentration. It is now realized the same GPCR can couple to differentGα proteins to result in “multifunctional signaling”. A criticalassumption of the GPCR multifunctional signaling theory is that aheterogeneity of active receptor conformations exists and that agonistligands differ in their ability to induce, stabilize, or select amongreceptor conformations, as described in the “stimulus trafficking”hypothesis. It follows that, upon binding, agonist ligand chemicalstructural parameters are among the most important determinants of GPCRconformation that influences type of Gα protein and signaling pathwayactivated.

A survey of 105 articles on the activity of 380 antagonists on 73biological G-protein-coupled receptor targets indicates that, in thissample dataset, 322 are inverse agonists and 58 (15%) are neutralantagonists. The predominance of inverse agonism agrees with theoreticalpredictions which indicate that neutral antagonists are the minorityspecies in pharmacological space (Kenakin, Mol Pharmacol.(2004);65:2-11).

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a neuropsychiatric disorder comprisingadministering to subject in need thereof a therapeutically effectiveamount of a compound capable of modulating 5-HT2 binding interactions.In one embodiment, the compound is capable of agonizing a 5-HT2c. Inanother embodiment, the compound is capable agonizing a 5-HT2c, whileantagonizing 5-HT2a and/or 5-HT2b.

In one aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a neuropsychiatric disorder. The methodincludes administering to a subject in need thereof a therapeuticallyeffective amount of a 5-HT2c agonizing compound.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a neuropsychiatric disorder. The methodincludes administering to a subject in need thereof a therapeuticallyeffective amount of a compound capable of modulating 5-HT2 bindinginteractions by directly modulating 5-HT2c, preferably selectivelyrelative to 5-HT2a and/or 5-HT2b.

In another embodiment, the invention provides a method of treating asubject suffering from or susceptible to a neuropsychiatric disorder.The method includes administering to a subject identified as in needthereof a therapeutically effective amount of a 5-HT2c agonizingcompound or a 5-HT2c selective compound.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a neuropsychiatric disorder, includingobesity, addiction, cocaine addiction, psychosis, anxiety, sleephomeostasis. The method includes administering to a subject agonizing5-HT2c.

In another aspect, the invention provides a method of treating a subjectsuffering from or susceptible to obesity, addiction, cocaine addiction,psychosis, anxiety comprising administering to the subject an effectiveamount of a compound capable of agonizing 5-HT2c (including selectivelyrelative to 5-HT2a and/or 5-HT2b), such that the subject is treated.

In one aspect, the invention provides a method of treating a subjectsuffering from or susceptible to a GPCR disorder comprisingadministering to subject in need thereof a therapeutically effectiveamount of a compound capable of modulating GPCR binding interactions. Inone embodiment, the compound is capable of agonizing a GPCR. In anotherembodiment, the compound is capable antagonizing a GPCR.

In another aspect, the compounds herein are functionally selectivecompounds that target serotonin histamine H₁, 5HT_(2A, 2B,2C), andacetylcholine muscarinic M₁-M₅ GPCRs. In aspects, the invention providesa method to selectively target serotonin histamine H₁, 5HT_(2A, 2B,2C),and acetylcholine muscarinic M₁-M₅ GPCRs in a subject comprisingadministering to the subject a compound herein.

In another aspect, the invention provides a method of treating orpreventing a GPCR-mediated disorder in a subject comprisingadministering to the subject identified as in need thereof a PATcompound. In certain embodiments, the PAT compound is a compound ofTable 1 (infra). In certain embodiments, the PAT compound is representedby the formula (I):

(I)

wherein,

R₁ is independently H, NH₂, NH(alkyl), N(alkyl)₂;

R₂ is independently —(CH₂)n-;

Each n is independently 1 or 2;

R₃ is independently H, OH, or halo;

R₄ is independently H, OH, or halo

Each R₅ is independently H, alkyl, or halo;

R₆ is independently H or alkyl; and

R₇ independently H, or N(alkyl)₂; or salt, hydrate or solvate thereof.

In certain embodiments, the PAT compound R₁ is —NMe₂. In certainembodiments, the PAT compound is(1R,3S)-(−)-Trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene.

In certain embodiments, the disorder is a neuropsychiatric disorder(e.g., obesity, addiction, anxiety, depression, schizophrenia, and sleepdisorders), a neurodegenerative disorder (e.g., Parkinson's Disease,Alzheimer's Disease), a neurological disorder (e.g., epilepsy), acardiovascular disorder (e.g., hypertension), a gastrointestinaldisorder (e.g., irritable bowel syndrome), or a genitor-urinary tractdisorder (e.g., bladder control). In certain embodiments, the disorderis cocaine addiction. In certain embodiments, the disorder is obesity.

In another aspect, the invention provides a method of inhibiting 5-HT2Cin a subject identified as in need of such treatment, comprisingadministering a PAT compound.

In another aspect, the invention provides a method of treating obesityin a subject, comprising administering to the subject identified as inneed thereof a PAT compound capable of selectively inhibiting the 5-HT2crelative to 5-HT2a or 5-HT2b. In certain embodiments, the bindinginteraction the for inhibiting 5-HT2c is at least 5-fold (alternativelyat least 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 500 fold) greaterthan for either 5-HT2a or 5-HT2b. In certain embodiments, the bindinginteraction for inhibiting 5-HT2c is at least 100-fold greater than foreither 5-HT2a or 5-HT2b.

In another aspect, the invention provides a method for identifying acompound that is capable of modulating 5-HT2c activity, comprising; (i)producing a three-dimensional representation of a molecule or molecularcomplex, wherein said molecule or molecular complex comprises a bindingpocket defined by structure coordinates of 5-HT2c; or b) athree-dimensional representation of a homologue of said molecule ormolecular complex, wherein said homologue comprises a binding pocketthat has a root mean square deviation from the backbone atoms of saidamino acids of not more than about 2.0 angstroms; (ii) producing athree-dimensional representation of a test compound; (iii) assessing thebinding interaction of the test compound with the target. In certainembodiments, the method further comprises contacting the test compoundwith a 5-HT2c and measuring the binding activity of the compound.

In another aspect, the invention provides a compound represented by theformula (I):

(I)

wherein,

R₁ is independently H, NH₂, NH(alkyl), N(alkyl)₂;

R₂ is independently —(CH₂)n-;

Each n is independently 1 or 2;

R₃ is independently H, OH, or halo;

R₄ is independently H, OH, or halo

Each R₅ is independently H, alkyl, or halo;

R₆ is independently H or alkyl; and

R₇ independently H, or N(alkyl)₂;

or salt, hydrate or solvate thereof.

In certain embodiments, the compound substituents at the 1-position andthe 3-position are in the trans-orientation to one another.

In another aspect, the invention provides a composition comprising acompound described herein (e.g., a compound of Formula (I)) and apharmaceutically acceptable carrier.

In another aspect, the invention provides a method making a compositioncomprising combining a compound described herein (e.g., a compound ofFormula (I)) and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a compound of the formula:

wherein,

R₁ is independently H, NH₂, NH(alkyl), N(alkyl)₂;

R₂ is independently —(CH₂)n-;

Each n is independently 1 or 2;

R₃ is independently H, OH, or halo;

R₄ is independently H, OH, or halo

Each R₅ is independently H, alkyl, or halo;

R₆ is independently H or alkyl;

R₇ independently H, N(alkyl)₂; and

R8 is independently aryl or heteroaryl, each optionally substituted with1-4 independent R₅;

or salt, hydrate or solvate thereof.

Results of structure-activity relationship (SAR) studies indicate thataffinity selectivity of the invention compounds for H₁ vs. 5HT_(2A) vs.5HT_(2B) vs. 5HT_(2C), vs, M₁ vs. M₂ vs. M₃ vs. M₄ vs. M₅ GPCRs isdependent on the stereochemistry of the substituents at the C1 (pendantphenyl or other aromatic) and C3 (amine) positions and, the chemicalnature of the C1 and C3 substituents, as well as, the chemicalsubstituents at the C6 and C7 positions of the tetrahydronapthalene ringsystem (carbon numbering as in Formula I, Table 1). Likewise, agonistvs. inverse agonist vs. antagonist activity at H₁ vs. 5HT_(2A) vs.5HT_(2B) vs. 5HT_(2C), vs, M₁ vs. M₂ vs. M₃ vs. M₄ vs. M₅ GPCRs isdetermined by the chemical nature and stereochemistry of thesubstituents(s) at C1, C3, C6, and C7. See, e.g., Bucholtz, E. C.,Wyrick, S. D., Owens, C. E., and Booth, R. G.1-Phenyl-3-dimethylaminotetralins (PATs): Effect of stereochemistry onbinding and function at brain histamine receptors. Medicinal ChemistryResearch 8:322-332 (1998).; Bucholtz, E. C., Brown., R. L., Tropsha, A.,Booth, R. G, and Wyrick, S. D. Synthesis, Evaluation and ComparativeMolecular Field Analysis of1-Phenyl-3-amino-1,2,3,4-tetrahydronaphthalenes as Ligands for HistamineH₁ Receptors. Journal of Medicinal Chemistry. 42:3041-3054(1999);Choksi, N. Y., Nix, William B., Wyrick, S. D., and Booth, R. G. A novelphenylaminotetralin recognizes histamine H₁ receptors and stimulatesdopamine synthesis in vivo in rat brain. Brain Research 852:151-160(2000); Booth R G, Moniri N H, Bakker R A, Choksi N Y, Timmerman H, andLeurs R. A novel phenylaminotetralin radioligand reveals asub-population of histamine H₁ receptors. Journal of Pharmacology andExperimental Therapeutics 302:328-336 (2002); Moniri N H,Covington-Strachan D, Booth R G. Ligand-directed functionalheterogeneity of histamine H₁ receptors: Novel dual-function ligandsselectively activate and block H₁-meditated phospholipas C and adenylylcyclase signaling. Journal of Pharmacology and ExperimentalTherapeutics, 311:274-281 (2004); Booth R G, Moniri N H. Ligand-directedmultifunctional signaling of histamine H₁ receptors InflammationResearch 54: S44-45 (2005); Ghoneim O M, Legere J A, Glbraikh A, TropshaA, Booth R G. Novel ligands for the human histamine H₁ receptor:Synthesis, pharmacology, and comparative molecular field analysisstudies of 2-dimethylamino-5-(6)-phenyl-1,2,3,4-tetrahydronaphthalenes.Bioorganic and Medicinal Chemistry, 14:6640-6658 (2006); Booth R G,Moniri N H. Novel Ligands Stabilize Stereo-Selective Conformations ofthe Histamine H1 Receptor to Activate Catecholamine Synthesis.Inflammation Research 56:1-12 (2007).

In another embodiment, the compounds herein can distinguish andselectively activate brain H₁ receptors that couple to the adenylylcyclase (AC)/cAMP vs. phospholipase C (PLC)/inositol phosphates (IP)intracellular signaling pathways to modulate brain catecholamine(dopamine, norepinephrine) neurotransmitter synthesis. In aspects, theinvention provides a method of selectively activating brain H₁ receptorsthat couple to the adenylyl cyclase (AC)/cAMP vs. phospholipase C(PLC)/inositol phosphates (IP) intracellular signaling pathways toaffect physiologically processes sensitive to H₁/AC/cAMP signaling,e.g., modulation brain catecholamine (dopamine, norepinephrine)neurotransmitter synthesis in a subject comprising administering to thesubject a compound herein.

In another aspect, the compounds herein are compounds that selectivelyenhance H₁-mediated AC/cAMP signaling to treat a patient suffering fromcertain neuropsychiatric diseases involving altered catecholamineneurotransmission. In aspects, the invention provides a method ofselectively enhancing H₁-mediated AC/cAMP signaling to treat a subjectsuffering from certain neuropsychiatric diseases involving alteredcatecholamine neurotransmission comprising administering to the subjecta compound herein.

In another embodiment, the compounds herein are compounds that areantagonists and inverse agonists of untoward H₁-mediated effects thatproceed via H₁/PLC/IP signaling, e.g., respiratory distress (bronchialconstriction), diarrhea (GI contractions), and edema and hypotension(increased vascular permeability), especially associated with theperipheral allergic response. In aspects, the invention provides amethod of antagonizing (e.g., blocking) untoward H₁-mediated effectsthat proceed via the PLC/IP pathway in a subject comprisingadministering to the subject a compound herein.

In another aspect, the PATs are antagonists as well as inverse agonistsat serotonin 5HT_(2A) and 5HT_(2B) receptors.

In another embodiment, the PATs are antagonists as well as inverseagonists at histamine H₁ receptors linked to PLC/IP signaling.

In another aspect, the PATs are antagonists as well as inverse agonistsat acetylcholine muscarinic M₁-M₅ receptors.

In another aspect, the PATs are antagonists as well as inverse agonistsand agonists at acetylcholine muscarinic M₁-M₅ receptors. In anotheraspect, the PATs are simultaneously inverse agonists at serotonin5HT_(2A) and 5HT_(2B) receptors and agonists at 5HT_(2C) receptors. Inaspects, the invention provides a method of treating or preventing adisease or disorder (e.g., psychiatric disorder; obesity) in a subjectcomprising administering to the subject a compound that issimultaneously an inverse agonist at serotonin 5HT_(2A) and 5HT_(2B)receptors and agonist at 5HT_(2C) receptors. In another embodiment, themethod is that wherein the subject is in need of treatment for both apsychiatric disorder and obesity.

In one embodiment, the compounds provide methods for pharmacologicallytreating a disease or disorder arising from disturbances in theacetylcholine muscarinic receptor system in a subject comprisingadministering to the subject a compound of any of the formulae herein.The compounds of any of the formulae herein havepharmacologically-relevant affinity for muscarinic M₁, M₂, M₃, M₄,and/or M₅ receptors and behave functionally as agonists, inverseagonists, and/or antagonists at one or more of the muscarinic receptors.

Typical diseases or disorders (Brown and Taylor, 2006, MuscarinicAgonists and Antagonists. In: Brunton L. L., Lazo, J. S., Parker, K. L.(Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics11^(th) Edition. McGraw-Hill, New York, N.Y., pp. 183-200) that respondto modulation of the pharmacology of muscarinic M₁, M₂, M₃, M₄, and/orM₅ receptors include but are not limited to: disorders of thegastrointestinal tract (constipation, diarrhea, excess acid,spasticity), urinary tract (frequency of urination, lack of urination,excess urination), glaucoma, asthma, Parkinson's disease, Alzheimer'sdisease, various disorders involving exocrine glands (problems withsweating, tear formation, saliva formation, mucous formation), andtreatment of poisoning from certain mushrooms (e.g., those containingnatural muscarine derivatives).

In another aspect, the invention provides a method for identifying acompound that modulates 5-HT2c, the method comprising obtaining acrystal structure of a 5-HT2c protein or obtaining information relatingto the crystal structure of a 5-HT2c protein and modeling a testcompound into or on the 5-HT2c protein structure to determine whetherthe compound modulates the interaction of a 5-HT2c protein. In certainembodiments, the step of modeling comprises modeling or determining theability of the compound to bind to or associate with a binding pocketdefined by structure coordinates of the one or more of transmembranedomains 1-7 of 5-HT2c.

Yet another aspect of the invention is a method for identifying acompound useful to treat or prevent obesity, addiction, cocaineaddiction, psychosis, anxiety. The method includes contacting a 5-HT2ccomplex with a test compound, and evaluating the ability of the testcompound to modulate (e.g., agonize or antagonize), 5-HT2c.

Yet another aspect of the invention is a method for identifying acompound that modulates the activity of 5-HT2c, the method comprisingusing the atomic coordinates of the one or more of transmembrane domains1-7 of 5-HT2c, to generate a three-dimensional structure (e.g., insilico) of a molecule comprising a binding pocket, and employing thethree-dimensional structure to identify a compound that modulates theactivity of the one or more of transmembrane domains 1-7 of 5-HT2c.

In another aspect, the invention provides a packaged compositionincluding a therapeutically effective amount of a 5-HT2c agonistcompound and a pharmaceutically acceptable carrier or diluent. Thecomposition may be formulated for treating a subject suffering from orsusceptible to a neuropsychiatric disorder (e.g., obesity), and packagedwith instructions to treat a subject suffering from or susceptible to aneuropsychiatric disorder.

In one aspect, the invention provides a kit for treating aneuropsychiatric disorder in a subject is provided and includes acompound herein, pharmaceutically acceptable esters, salts, and prodrugsthereof, and instructions for use. In further aspects, the inventionprovides kits for agonizing 5-HT2c, assessing the efficacy of ananti-obesity treatment in a subject, monitoring the progress of asubject being treated with a 5-HT2c agonist, selecting a subject with aneuropsychiatric disorder for treatment with 5-HT2c agonist, and/ortreating a subject suffering from or susceptible to a neuropsychiatricdisorder (e.g., obesity). In certain embodiments, the inventionprovides: a kit for treating a neuropsychiatric disorder in a subject,the kit comprising a compound capable of modulating (e.g., agonizing)5-HT2c agonist activity. In other aspects the compound selectivelyagonizes 5-HT2c relative to 5-HT2a and/or 5-HT2b. In other aspects thecompound selectively antagonizes 5-HT2a and/or 5-HT2b.

In another aspect, the invention relates to a three-dimensionalstructure of a one or more of transmembrane domains 1-7 of 5-HT2c, eachalone or combinations thereof.

Thus, the present invention provides molecules or molecular complexesthat comprise either one or both of these binding pockets or homologuesof either binding pocket that have similar three-dimensional shapes.

The invention also provides a pharmaceutical composition of thecompounds described herein, comprising a compound capable of agonizing5-HT2c; a compound capable of agonizing 5-HT2c selectively relative to5-HT2a and/or 5-HT2b; a compound capable of agonizing 5-HT2c andantagonizing 5-HT2a and/or 5-HT2b; or a pharmaceutically acceptableester, salt, or prodrug thereof, together with a pharmaceuticallyacceptable carrier.

In another aspect, the invention provides a machine readable storagemedium which comprises the structural coordinates of a binding pocketdefining the one or more of transmembrane domains 1-7 of 5-HT2c.

In another aspect, the invention provides a computer for producing athree-dimensional representation of a molecule or molecular complex,wherein said molecule or molecular complex comprises a binding pocketdefined by structure coordinates of the one or more of transmembranedomains 1-7 of 5-HT2c; or b) a three-dimensional representation of ahomologue of said molecule or molecular complex, wherein said homologuecomprises a binding pocket that has a root mean square deviation fromthe backbone atoms of said amino acids of not more than about 2.0angstroms. The computer includes: (i) a machine-readable data storagemedium comprising a data storage material encoded with machine-readabledata, wherein said data comprises the structure coordinates of the oneor more of transmembrane domains 1-7 of 5-HT2c; (ii) a working memoryfor storing instructions for processing said machine-readable data;(iii) a central-processing unit coupled to said working memory and tosaid machine-readable data storage medium for processing said machinereadable data into said three-dimensional representation; and (iv) adisplay coupled to said central-processing unit for displaying saidthree-dimensional representation.

The invention also provides methods for designing, evaluating andidentifying compounds which bind to the aforementioned binding pockets.Other embodiments of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described below with reference to thefollowing non-limiting examples and with reference to the followingfigures, in which:

FIG. 1. depicts stereochemical relationship of cis- andtrans-1-phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (PAT)compounds.

FIG. 2. depicts (2A-2C) representative binding curves for[³H]-ketanserin labeled 5HT_(2A) receptors and [³H]-mesulergine labeled5-HT_(2B) receptors and 5-HT_(2C) receptors.

FIG. 3. depicts representative 5-HT_(2A), 5-HT_(2B), and 5-HT_(2C)radioligand displacement curves for (−)-trans-PAT.

FIG. 4. depicts assessment of (−)-trans-PAT agonist activity at5HT₂-subtype receptors.

FIG. 5. depicts assessment of the ability of (−)-trans-PAT to act as a5HT_(2A) and 5HT_(2B) receptor antagonist regarding 5-HT-mediatedstimulation of PLC/[³H]-IP formation.

FIG. 6. (Example 9, 1(c) FIG. 2) depicts curves for 5-HT_(2A) binding

FIG. 7. (Example 9, 1(c) FIG. 3) depicts curves for 5-HT_(2B) binding.

FIG. 8. (Example 9, 1(c) FIG. 4) depicts curves for 5-HT_(2C) binding.

FIG. 9. (Example 9, 1(d) FIG. 5) depicts curves for 5-HT_(2C) binding.

FIG. 10. (Example 9, 1(d) FIG. 6) depicts curves for 5-HT_(2A) binding.

FIG. 11. (Example 9, 1(d) FIG. 7) depicts curves for 5-HT_(2C) binding.

FIG. 12. (Example 10, 2(a) FIG. 9) depicts curves for phospholipase C(PLC) activity.

FIG. 13. (Example 10, 2(a) FIG. 10) depicts curves for inositolphosphates (IP) activity.

FIG. 14. (Example 10, 2(a) FIG. 11) depicts curves for PLC activity.

FIG. 15. (Example 10, 2(a) FIG. 12) depicts assessment for PLC activity.

FIG. 16. (Example 10, 2(a) FIG. 13) depicts assessment for PLC activity.

FIG. 17. (Example 11, 2 FIG. 16) depicts PATS having fixed pendantphenyl rings.

FIG. 18. depicts (−)-Trans-PAT competitive antagonism of 5-HT activationof 5-HT_(2A) receptors.

FIG. 19. depicts (−)-Trans-PAT inverse agonist activity at serotonin5-HT2_(A) receptors.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have now discovered a therapeutic strategy thataddresses selective disease treatment and prevention (i.e., havingreduced or minimized adverse side effects) by selectively targeting5-HT2c. Such interactions are relevant for modulation of 5-HT2c mediateddisorders, particularly in certain neuropsychiatric disorder types where5-HT2 mechanisms play a significant role.

The present invention relates, at least in part, to the discovery thatthe 5-HT2c interactions are useful as targets (e.g., selective) forneuropsychiatric disorder therapy.

1. Definitions

Before further description of the present invention, and in order thatthe invention may be more readily understood, certain terms are firstdefined and collected here for convenience.

The term “administration” or “administering” includes routes ofintroducing the compound of the invention(s) to a subject to performtheir intended function. Examples of routes of administration that maybe used include injection (subcutaneous, intravenous, parenterally,intraperitoneally, intrathecal), oral, inhalation, rectal andtransdermal. The pharmaceutical preparations may be given by formssuitable for each administration route. For example, these preparationsare administered in tablets or capsule form, by injection, inhalation,eye lotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administration is preferred. The injection can bebolus or can be continuous infusion. Depending on the route ofadministration, the compound of the invention can be coated with ordisposed in a selected material to protect it from natural conditionswhich may detrimentally effect its ability to perform its intendedfunction. The compound of the invention can be administered alone, or inconjunction with either another agent as described above or with apharmaceutically-acceptable carrier, or both. The compound of theinvention can be administered prior to the administration of the otheragent, simultaneously with the agent, or after the administration of theagent. Furthermore, the compound of the invention can also beadministered in a pro-drug form which is converted into its activemetabolite, or more active metabolite in vivo.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. The term alkyl further includesalkyl groups, which can further include oxygen, nitrogen, sulfur orphosphorus atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen, sulfur or phosphorus atoms. Inpreferred embodiments, a straight chain or branched chain alkyl has 30or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain,C₃-C₃₀ for branched chain), preferably 26 or fewer, and more preferably20 or fewer, and still more preferably 4 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 3, 4, 5, 6 or 7 carbons in the ring structure.

Moreover, the term alkyl as used throughout the specification andsentences is intended to include both “unsubstituted alkyls” and“substituted alkyls,” the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. Cycloalkyls can be further substituted, e.g., with thesubstituents described above. An “alkylaryl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl”also includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six, and still morepreferably from one to four carbon atoms in its backbone structure,which may be straight or branched-chain. Examples of lower alkyl groupsinclude methyl, ethyl, n-propyl, i-propyl, tert-butyl, hexyl, heptyl,octyl and so forth. In preferred embodiment, the term “lower alkyl”includes a straight chain alkyl having 4 or fewer carbon atoms in itsbackbone, e.g., C1-C4 alkyl.

The terms “alkoxyalkyl,” “polyaminoalkyl” and “thioalkoxyalkyl” refer toalkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond,respectively. For example, the invention contemplates cyano andpropargyl groups.

The term “aryl” as used herein, refers to the radical of aryl groups,including 5- and 6-membered single-ring aromatic groups that may includefrom zero to four heteroatoms, for example, benzene, pyrrole, furan,thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Aryl groups also include polycyclic fused aromatic groups such asnaphthyl, quinolyl, indolyl, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles,” “heteroaryls” or “heteroaromatics.” The aromatic ring canbe substituted at one or more ring positions with such substituents asdescribed above, as for example, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato,cyano, amino (including alkyl amino, dialkylamino, arylamino,diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Arylgroups can also be fused or bridged with alicyclic or heterocyclic ringswhich are not aromatic so as to form a polycycle (e.g., tetralin).

The term “associating with” refers to a condition of proximity between achemical entity or compound, or portions thereof, and a binding pocketor binding site on a protein. The association may be non-covalent(wherein the juxtaposition is energetically favored by hydrogen bondingor van der Waals or electrostatic interactions) or it may be covalent.

The term “binding pocket”, as used herein, refers to a region of amolecule or molecular complex, that, as a result of its shape, favorablyassociates with another chemical entity or compound.

The language “biological activities” of a compound of the inventionincludes all activities elicited by compound of the inventions in aresponsive cell. It includes genomic and non-genomic activities elicitedby these compounds.

“Biological composition” or “biological sample” refers to a compositioncontaining or derived from cells or biopolymers. Cell-containingcompositions include, for example, mammalian blood, red cellconcentrates, platelet concentrates, leukocyte concentrates, blood cellproteins, blood plasma, platelet-rich plasma, a plasma concentrate, aprecipitate from any fractionation of the plasma, a supernatant from anyfractionation of the plasma, blood plasma protein fractions, purified orpartially purified blood proteins or other components, serum, semen,mammalian colostrum, milk, saliva, placental extracts, acryoprecipitate, a cryosupernatant, a cell lysate, mammalian cellculture or culture medium, products of fermentation, ascites fluid,proteins induced in blood cells, and products produced in cell cultureby normal or transformed cells (e.g., via recombinant DNA or monoclonalantibody technology). Biological compositions can be cell-free. In apreferred embodiment, a suitable biological composition or biologicalsample is a red blood cell suspension. In some embodiments, the bloodcell suspension includes mammalian blood cells. Preferably, the bloodcells are obtained from a human, a non-human primate, a dog, a cat, ahorse, a cow, a goat, a sheep or a pig. In preferred embodiments, theblood cell suspension includes red blood cells and/or platelets and/orleukocytes and/or bone marrow cells.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “diastereomers” refers to stereoisomers with two or morecenters of dissymmetry and whose molecules are not mirror images of oneanother.

The term “effective amount” includes an amount effective, at dosages andfor periods of time necessary, to achieve the desired result, e.g.,sufficient to treat a disorder delineated herein. An effective amount ofcompound of the invention may vary according to factors such as thedisease state, age, and weight of the subject, and the ability of thecompound of the invention to elicit a desired response in the subject.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. An effective amount is also one in which any toxic ordetrimental effects (e.g., side effects) of the compound of theinvention are outweighed by the therapeutically beneficial effects.

A therapeutically effective amount of compound of the invention (i.e.,an effective dosage) may range from about 0.001 to 30 mg/kg body weight,preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Theskilled artisan will appreciate that certain factors may influence thedosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a compound of the invention can include a singletreatment or, preferably, can include a series of treatments. In oneexample, a subject is treated with a compound of the invention in therange of between about 0.1 to 20 mg/kg body weight, one time per weekfor between about 1 to 10 weeks, preferably between 2 to 8 weeks, morepreferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. It will also be appreciated that the effectivedosage of a compound of the invention used for treatment may increase ordecrease over the course of a particular treatment.

The term “enantiomers” refers to two stereoisomers of a compound whichare non-superimposable mirror images of one another. An equimolarmixture of two enantiomers is called a “racemic mixture” or a“racemate.”

The term “haloalkyl” is intended to include alkyl groups as definedabove that are mono-, di- or polysubstituted by halogen, e.g.,fluoromethyl and trifluoromethyl.

The terms “halogen” and “halo” designate —F, —Cl, —Br or —I.

The term “hydroxyl” means —OH.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,sulfur and phosphorus.

The term “homeostasis” is art-recognized to mean maintenance of static,or constant, conditions in an internal environment.

The language “improved biological properties” refers to any activityinherent in a compound of the invention that enhances its effectivenessin vivo. In a preferred embodiment, this term refers to any qualitativeor quantitative improved therapeutic property of a compound of theinvention, such as reduced toxicity.

The term “GPCR disorder” includes any disease, disorder or symptomsthereof that are mediated by a G protein-coupled receptor (e.g., 5-HT2a,5-HT2b, 5-HT2c, muscarinic M1-M5). Diseases and disorders mediated bysuch GPCRs include, for example, neuropsychiatric disorders (e.g.,obesity, addiction, cocaine addiction, psychosis anxiety, depression,schizophrenia, psychosis, and sleep disorders), neurodegenerativedisorders (e.g., Parkinson's Disease, Alzheimer's Disease), neurologicaldisorders (e.g., epilepsy), cardiovascular disorders (e.g.,hypertension), gastrointestinal disorders (e.g., irritable bowelsyndrome), and genitor-urinary tract disorders (e.g., bladder control).

The language “M1-M5 GPCR” refers to the cholinergic muscarinic M1-M5neurotransmitter G protein-coupled receptors (including those delineatedherein) that.

The language “5-HT2” refers to the serotonin receptors (including thosedelineated herein) such as 5-HT2a, 5-HT2b and 5-HT2c sub-types.

The term “optionally substituted” is intended to encompass groups thatare unsubstituted or are substituted by other than hydrogen at one ormore available positions, typically 1, 2, 3, 4 or 5 positions, by one ormore suitable groups (which may be the same or different). Such optionalsubstituents include, for example, hydroxy, halogen, cyano, nitro,C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, C₁-C₈alkoxy, C₂-C₈alkyl ether,C₃-C₈alkanone, C₁-C₈alkylthio, amino, mono- or di-(C1-C₈alkyl)amino,haloC₁-C₈alkyl, haloC₁-C₈alkoxy, C₁-C₈alkanoyl, C₂-C₈alkanoyloxy,C₁-C₈alkoxycarbonyl, —COOH, —CONH₂, mono- ordi-(C₁-C₈alkyl)aminocarbonyl, —SO₂NH₂, and/or mono ordi(C₁-C₈alkyl)sulfonamido, as well as carbocyclic and heterocyclicgroups. Optional substitution is also indicated by the phrase“substituted with from 0 to X substituents,” where X is the maximumnumber of possible substituents. Certain optionally substituted groupsare substituted with from 0 to 2, 3 or 4 independently selectedsubstituents (i.e., are unsubstituted or substituted with up to therecited maximum number of substituents).

The term “isomers” or “stereoisomers” refers to compounds which haveidentical chemical constitution, but differ with regard to thearrangement of the atoms or groups in space.

The term “modulate” refers to an increase or decrease, e.g., in theability of a compound inhibit activity of a target in response toexposure to a compound of the invention, including for example in ansubject (e.g., animal, human) such that a desired end result isachieved, e.g., a therapeutic result.

The term “obtaining” as in “obtaining a compound capable of modulating(agonizing, antagonizing) a target delineated herein and is intended toinclude purchasing, synthesizing or otherwise acquiring the compound.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The terms “polycyclyl” or “polycyclic radical” refer to the radical oftwo or more cyclic rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle can be substituted with suchsubstituents as described above, as for example, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “prodrug” or “pro-drug” includes compounds with moieties thatcan be metabolized in vivo. Generally, the prodrugs are metabolized invivo by esterases or by other mechanisms to active drugs. Examples ofprodrugs and their uses are well known in the art (See, e.g., Berge etal. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). The prodrugscan be prepared in situ during the final isolation and purification ofthe compounds, or by separately reacting the purified compound in itsfree acid form or hydroxyl with a suitable esterifying agent. Hydroxylgroups can be converted into esters via treatment with a carboxylicacid. Examples of prodrug moieties include substituted andunsubstituted, branch or unbranched lower alkyl ester moieties, (e.g.,propionoic acid esters), lower alkenyl esters, di-lower alkyl-aminolower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino loweralkyl esters (e.g., acetyloxymethyl ester), acyloxy lower alkyl esters(e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-loweralkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halo,or methoxy substituents) aryl and aryl-lower alkyl esters, amides,lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Preferredprodrug moieties are propionoic acid esters and acyl esters. Prodrugswhich are converted to active forms through other mechanisms in vivo arealso included.

The language “a prophylactically effective amount” of a compound refersto an amount of a compound of the invention any formula herein orotherwise described herein which is effective, upon single or multipledose administration to the patient, in preventing or treating a disorderherein.

The language “reduced toxicity” is intended to include a reduction inany undesired side effect elicited by a compound of the invention whenadministered in vivo.

The term “sulfhydryl” or “thiol” means —SH.

The term “subject” includes organisms which are capable of sufferingfrom a disorder herein or who could otherwise benefit from theadministration of a compound of the invention of the invention, such ashuman and non-human animals. Preferred humans include human patientssuffering from or prone to suffering from a neuropsychiatric disorder orassociated state, as described herein. The term “non-human animals” ofthe invention includes all vertebrates, e.g., mammals, e.g., rodents,e.g., mice, and non-mammals, such as non-human primates, e.g., sheep,dog, cow, chickens, amphibians, reptiles, etc.

The term “susceptible to a neuropsychiatric disorder” is meant toinclude subjects at risk of developing a neuropsychiatric disorder,e.g., including those delineated herein, i.e., subjects suffering from aneuropsychiatric disorder or symptom thereof, subjects having a familyor medical history of neuropsychiatric disorder or symptom thereof, andthe like.

The phrases “systemic administration,” “administered systemically”,“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound of the invention(s), drugor other material, such that it enters the patient's system and, thus,is subject to metabolism and other like processes, for example,subcutaneous administration.

The language “therapeutically effective amount” of a compound of theinvention of the invention refers to an amount of an agent which iseffective, upon single or multiple dose administration to the patient,treating or preventing a neuropsychiatric disorder and/or symptoms of aneuropsychiatric disorder, or in prolonging the survivability of thepatient with such a neuropsychiatric disorder beyond that expected inthe absence of such treatment.

With respect to the nomenclature of a chiral center, terms “d” and “l”configuration are as defined by the IUPAC Recommendations. As to the useof the terms, diastereomer, racemate, epimer and enantiomer will be usedin their normal context to describe the stereochemistry of preparations.

2. Compounds of the Invention

In one aspect, the invention provides compounds capable of modulating(e.g., inhibiting or stimulating) (directly or indirectly) 5-HT bindingactivity.

In one embodiment, the invention provides a compound capable ofagonizing 5-HT2c; and pharmaceutically acceptable esters, salts, andprodrugs thereof. In certain embodiments, the compound is a compound ofFormula (I).

Certain preferred compounds include compounds specifically delineatedherein:

TABLE 1 Compounds: Formula I

Configuration is stereochemistry at Cl & C3 PAT # Config R₁ R₂ R₃ R₄ R₅R₆ R₇ 1 (1R,3S)- N(CH₃)₂ CH₂ H H H H H (−)-trans 2 (1S,3R)- N(CH₃)₂ CH₂H H H H H (+)-trans 3 (1R,3R)- N(CH₃)₂ CH₂ H H H H H (−)-cis 4 (1S,3S)-N(CH₃)₂ CH₂ H H H H H (+)-cis 5 (±)-trans N(CH₃)₂ (CH₂)₂ H H H H H (PAB)6 (±)-cis N(CH₃)₂ (CH₂)₂ H H H H H (PAB) 7 (±)-trans N(CH₃)₂ CH₂ Cl OH HH H 8 (±)-cis N(CH₃)₂ CH₂ Cl OH H H H 9 (±)-trans N(CH₃)₂ CH₂ OH OH H HH 10 (±)-cis N(CH₃)₂ CH₂ OH OH H H H 11 (±)-trans N(CH₃)₂ CH₂ H H H CH₃H 12 (±)-cis N(CH₃)₂ CH₂ H H H CH₃ H 13 (±)-trans N(CH₃)₂ CH₂ H H p-Cl HH 14 (±)-trans N(CH₃)₂ CH₂ H H p-F H H 15 (±)-trans N(CH₃)₂ CH₂ H H pCH₃H H 16 (±)-trans N(CH₃)₂ CH₂ H H o-Cl H H 17 (±)-trans N(CH₃)₂ CH₂ H HoCH₃ H H 18 (±)-trans N(CH₃)₃ CH₂ H H H H H 19 (±)-cis N(CH₃)₃ CH₂ H H HH H 20 (±)-trans NH(CH₃) CH₂ H H H H H 21 (±)-cis NH(CH₃) CH₂ H H H H H22 (±)-trans NH₂ CH₂ H H H H H 23 (±)-cis NH₂ CH₂ H H H H H 24 (±)-transNH₂ CH₂ OH OH H H H 25 (±)-cis NH₂ CH₂ OH OH H H H 26 (±)-trans H CH₂ HH H H N(CH₃) 27 (±)cis H CH₂ H H H H N(CH₃) 28 (±)-trans N(C₂H₅)₂ CH₂ HH H H H 29 (±)-trans N(C₃H₅)₂ CH₂ H H H H H 30 (±)-trans NCH₃(C₃H₅) CH₂H H H H H 31 (±)-trans NH(C₃H₅) CH₂ H H H H HSynthesis of New PAT Analogs with Changes to the C(1) Pendant PhenylSubstituent

Based on binding, function, 3D QSAR, and molecular modeling results inPreliminary Data, we demonstrate that the (−)-trans-PAT C(1) pendantphenyl moiety is critical to providing full-efficacy 5HT_(2C) agonistactivity without activation of 5HT_(2A) and 5HT_(2B) receptors. TestingPAT pendant phenyl ring substitution and orientation will help todetermine optimal steric and electrostatic binding interactions with5HT₂ active site amino acids to obtain 5HT_(2C) agonists and/or5HT_(2A)/5HT_(2B) antagonists with higher affinity, potency, and/orselectivity.

TABLE 2 Compounds

Chart of New C(1) Substituted PAT Analogs

The invention also relates to the pharmaceutically acceptable salts andesters of the above-mentioned compounds.

Naturally occurring or synthetic isomers can be separated in severalways known in the art. Methods for separating a racemic mixture of twoenantiomers include chromatography using a chiral stationary phase (see,e.g., “Chiral Liquid Chromatography,” W. J. Lough, Ed. Chapman and Hall,New York (1989)). Enantiomers can also be separated by classicalresolution techniques. For example, formation of diastereomeric saltsand fractional crystallization can be used to separate enantiomers. Forthe separation of enantiomers of carboxylic acids, the diastereomericsalts can be formed by addition of enantiomerically pure chiral basessuch as brucine, quinine, ephedrine, strychnine, and the like.Alternatively, diastereomeric esters can be formed with enantiomericallypure chiral alcohols such as menthol, followed by separation of thediastereomeric esters and hydrolysis to yield the free, enantiomericallyenriched carboxylic acid. For separation of the optical isomers of aminocompounds, addition of chiral carboxylic or sulfonic acids, such ascamphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid canresult in formation of the diastereomeric salts.

According to another embodiment, the invention provides compounds whichassociate with or bind to GPCR or binding pocket thereof produced oridentified by the methods described herein.

3. Uses of the Compounds of the Invention

In one embodiment, the invention provides methods for treating a subjectfor a GPCR-mediated disorder disorder, by administering to the subjectan effective amount of a compound capable of modulating (agonizing,antagonizing) a GPCR target. A GPCR disorder includes diseases anddisorders mediated by such GPCRs. The herein delineated compounds,compositions and methods are useful for treating or preventing disorderincluding, for example, neuropsychiatric disorders (e.g., obesity,addiction, anxiety, depression, schizophrenia, and sleep disorders),neurodegenerative disorders (e.g., Parkinson's Disease, Alzheimer'sDisease), neurological disorders (e.g., epilepsy), cardiovasculardisorders (e.g., 5-HT2b mediated disease, hypertension),gastrointestinal disorders (e.g., irritable bowel syndrome), andgenitor-urinary tract disorders (e.g., bladder control). In certainembodiments, the subject is a mammal, e.g., a primate, e.g., a human.

In one embodiment, the invention provides compounds and methods fortreating a subject for a histamine (e.g., H1, H2, H3, H4)-mediateddisorder, by administering to the subject an effective amount of acompound capable of modulating (agonizing, antagonizing) a histaminetarget. A histamine disorder includes diseases and disorders mediated bysuch histamine (e.g., H1). The herein delineated compounds, compositionsand methods are useful for treating or preventing disorder including,for example, respiratory distress (e.g., bronchial constriction),diarrhea (GI contractions), edema, and hypotension (e.g., increasedvascular permeability), allergic response, and neuropsychiatric,neurodegenerative and neurological disorders herein.

In this embodiment, the compounds of the invention may either directlyor indirectly modulate (e.g., agonize, stimulate) the activity of 5-HT2cor specific domains thereof. A cell can be contacted with a compound ofthe invention to agonize 5-HT2c and modulate 5-HT2c mediated activity.Contacting cells or administering the compounds of the invention to asubject is one method of treating a cell or a subject suffering from orsusceptible to unwanted or undesired 5-HT2C mediated activity or a5-HT2c mediated disorder.

In one embodiment, a method of treating a subject suffering from orsusceptible to a 5-HT2c disorder includes administering to a subject inneed thereof a therapeutically effective amount of a compound capable ofdirectly or indirectly modulating the activity of 5-HT2c, to therebytreat the subject. Exemplary compounds include those compounds describedherein (e.g., PAT, etc.).

Thus, in one embodiment, the invention provides methods for treating asubject for a 5-HT2C disorder, by administering to the subject aneffective amount of a compound capable of agonizing 5-HT2c.

In certain embodiments, the methods of the invention includeadministering to a subject a therapeutically effective amount of acompound of the invention in combination with another pharmaceuticallyactive compound. Other pharmaceutically active compounds that may beused can be found in Harrison's Principles of Internal Medicine,Thirteenth Edition, Eds. T. R. Harrison et al. McGraw-Hill N.Y., N.Y.;and the Physicians Desk Reference 50th Edition 1997, Oradell N.J.,Medical Economics Co., the complete contents of which are expresslyincorporated herein by reference. The compound of the invention and thepharmaceutically active compound may be administered to the subject inthe same pharmaceutical composition or in different pharmaceuticalcompositions (at the same time or at different times).

In certain embodiments, the compound of the invention can be used incombination therapy with conventional anti-obesity, (e.g., fatabsorption blockers). Certain 5-HT drugs (e.g., 5-HTa or 5-HTbantagonists) have an undesirable side effect profile that tend to makethem less then optimal or unsuitable for certain patients, that is, theydemonstrate cardiovascular (e.g., valvular heart disease, pulmonaryhypertension, cardiotoxicity) or psychiatric undesirable and or lifethreatening side effect profiles.

Determination of a therapeutically effective amount or aprophylactically effective amount of the compound of the invention ofthe invention, can be readily made by the physician or veterinarian (the“attending clinician”), as one skilled in the art, by the use of knowntechniques and by observing results obtained under analogouscircumstances. The dosages may be varied depending upon the requirementsof the patient in the judgment of the attending clinician; the severityof the condition being treated and the particular compound beingemployed. In determining the therapeutically effective amount or dose,and the prophylactically effective amount or dose, a number of factorsare considered by the attending clinician, including, but not limitedto: the specific 5-HT2c disorder involved; pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the desired time course of treatment; the species ofmammal; its size, age, and general health; the specific diseaseinvolved; the degree of or involvement or the severity of the disease;the response of the individual patient; the particular compoundadministered; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the kind of concurrent treatment (i.e., the interaction of thecompound of the invention with other co-administered therapeutics); andother relevant circumstances.

Treatment can be initiated with smaller dosages, which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum effect under the circumstances isreached. For convenience, the total daily dosage may be divided andadministered in portions during the day if desired. A therapeuticallyeffective amount and a prophylactically effective amount of a compoundof the invention of the invention is expected to vary from about 0.1milligram per kilogram of body weight per day (mg/kg/day) to about 100mg/kg/day.

Compounds determined to be effective for the prevention or treatment of5-HT2c disorders in animals, e.g., dogs, chickens, and rodents, may alsobe useful in treatment of 5-HT2c disorders in humans. Those skilled inthe art of treating 5-HT2c disease in humans will know, based upon thedata obtained in animal studies, the dosage and route of administrationof the compound to humans. In general, the dosage and route ofadministration in humans is expected to be similar to that in animals.

The identification of those patients who are in need of prophylactictreatment for 5-HT2C disorders is well within the ability and knowledgeof one skilled in the art. Certain of the methods for identification ofpatients which are at risk of developing 5-HT2C disorders which can betreated by the subject method are appreciated in the medical arts, suchas family history, and the presence of risk factors associated with thedevelopment of that disease state in the subject patient. A clinicianskilled in the art can readily identify such candidate patients, by theuse of, for example, clinical tests, physical examination andmedical/family history.

A method of assessing the efficacy of a treatment in a subject includesdetermining the pre-treatment extent of a 5-HT2C disorder by methodswell known in the art (e.g., determining level of markers for the 5-HT2Cdisorder) and then administering a therapeutically effective amount of acompound delineated herein according to the invention to the subject.After an appropriate period of time after the administration of thecompound (e.g., 1 day, 1 week, 2 weeks, one month, six months), theextent of the 5-HT2C disorder is determined again. The modulation (e.g.,decrease) of the extent or invasiveness of the 5-HT2C disorder indicatesefficacy of the treatment. The extent or invasiveness of the 5-HT2Cdisorder may be determined periodically throughout treatment. Forexample, the extent or invasiveness of the 5-HT2C disorder may bechecked every few hours, days or weeks to assess the further efficacy ofthe treatment. A decrease in extent or invasiveness of the 5-HT2Cdisorder indicates that the treatment is efficacious. The methoddescribed may be used to screen or select patients that may benefit fromtreatment with a modulating compound of a 5-HT2C disorder.

As used herein, “obtaining a biological sample from a subject,” includesobtaining a sample for use in the methods described herein. A biologicalsample is described above.

Yet another aspect presents a method to identify a compound thatmodulates the interaction of 5-HT2C or specific domains thereof. Themethod may include obtaining the crystal structure of 5-HT2C or specificdomains thereof (optionally apo form or complexed) or obtaining theinformation relating to the crystal structure of 5-HT2C or specificdomains thereof (optionally apo form or complexed), in the presenceand/or absence of the test compound. Compounds may then be computermodeled into or on the 5-HT2C structure, or specific domains thereof(e.g., a binding site of the crystal structure) to predict stabilizationof the interaction between the 5-HT2C or specific domains thereof andthe test compound. Once potential modulating compounds are identified,the compounds may be screened using cellular assays, such as the onesidentified herein and competition assays known in the art. Compoundsidentified in this manner are useful as therapeutic agents.

In another aspect, a compound of the invention is packaged in atherapeutically effective amount with a pharmaceutically acceptablecarrier or diluent. The composition may be formulated for treating asubject suffering from or susceptible to a 5-HT2C disorder, and packagedwith instructions to treat a subject suffering from or susceptible to a5-HT2C disorder.

In another aspect, the invention provides methods for modulating 5-HT2Cdisease. In one embodiment, a method of modulating 5-HT2C (or a 5-HT2Cdisorder) according to the invention includes contacting cells with acompound capable of modulating 5-HT2C (or a 5-HT2C disorder), orspecific domains thereof. In either embodiment, the contacting may be invitro, e.g., by addition of the compound to a fluid surrounding thecells, for example, to the growth media in which the cells are living orexisting. The contacting may also be by directly contacting the compoundto the cells. Alternately, the contacting may be in vivo, e.g., bypassage of the compound through a subject; for example, afteradministration, depending on the route of administration, the compoundmay travel through the digestive tract or the blood stream or may beapplied or administered directly to cells in need of treatment.

In another aspect, methods of inhibiting a 5-HT2C disorder in a subjectinclude administering an effective amount of a compound of the invention(i.e., a compound described herein) to the subject. The administrationmay be by any route of administering known in the pharmaceutical arts.The subject may have a 5-HT2C disorder, may be at risk of developing a5-HT2C disorder, or may need prophylactic treatment prior to anticipatedor unanticipated exposure to a conditions capable of increasingsusceptibility to a 5-HT2C disorder.

In one aspect, a method of monitoring the progress of a subject beingtreated with a compound herein includes determining the pre-treatmentstatus (e.g., progression, target profile, Marker profile) of the 5-HT2Cdisorder, administering a therapeutically effective amount of a compoundherein to the subject, and determining the status (e.g., progression,target profile, Marker profile) of the 5-HT2C disorder after an initialperiod of treatment with the compound, wherein the modulation of thestatus indicates efficacy of the treatment.

The subject may be at risk of a 5-HT2C disorder, may be exhibitingsymptoms of a 5-HT2C disorder, may be susceptible to a 5-HT2C disorderand/or may have been diagnosed with a 5-HT2C disorder.

If the modulation of the status indicates that the subject may have afavorable clinical response to the treatment, the subject may be treatedwith the compound. For example, the subject can be administeredtherapeutically effective dose or doses of the compound.

In another aspect, methods for evaluating a test compound comprisecontacting a 5-HT2C or specific domains thereof with a test compound(complex), and evaluating the binding interaction following contact,wherein a change in the stability of the complex relative to a referencevalue is an indication that the test compound modulates the stability ofthe complex.

The 5-HT2C or specific domains thereof complex may be modeled in silico,or may be a complex within a cell, isolated from a cell, recombinantlyexpressed, purified or isolated from a cell or recombinant expressionsystem or partially purified or isolated from a cell or recombinantexpression system.

Kits of the invention include kits for treating a 5-HT2C disorder in asubject. The kit may include a compound of the invention, for example, acompound described herein, pharmaceutically acceptable esters, salts,and prodrugs thereof, and instructions for use. The instructions for usemay include information on dosage, method of delivery, storage of thekit, etc. The kits may also include, reagents, for example, testcompounds, buffers, media (e.g., cell growth media), cells, etc. Testcompounds may include known compounds or newly discovered compounds, forexample, combinatorial libraries of compounds. One or more of the kit ofthe invention may be packaged together, for example, a kit for assessingthe efficacy of an treatment for a 5-HT2C disorder may be packaged witha kit for monitoring the progress of a subject being treated for a5-HT2C disorder according to the invention.

The present methods can be performed on cells in culture, e.g. in vitroor ex vivo, or on cells present in an animal subject, e.g., in vivo.Compounds of the inventions can be initially tested in vitro usingprimary cultures of cells, e.g., transformed cells, and the like.

The present method can be performed on cells in culture, e.g. in vitroor ex vivo, or on cells present in an animal subject, e.g., in vivo.Compound of the invention can be initially tested in vitro using cellsfrom the respiratory tract from embryonic rodent pups (See e.g. U.S.Pat. No. 5,179,109—fetal rat tissue culture), or other mammalian (Seee.g. U.S. Pat. No. 5,089,517—fetal mouse tissue culture) ornon-mammalian animal models.

Alternatively, the effects of compound of the invention can becharacterized in vivo using animals models.

4. Pharmaceutical Compositions

The invention also provides a pharmaceutical composition, comprising aneffective amount of a compound of the and a pharmaceutically acceptablecarrier. In a further embodiment, the effective amount is effective totreat a 5-HT2C disorder, as described previously.

In an embodiment, the compound of the invention is administered to thesubject using a pharmaceutically-acceptable formulation, e.g., apharmaceutically-acceptable formulation that provides sustained deliveryof the compound of the invention to a subject for at least 12 hours, 24hours, 36 hours, 48 hours, one week, two weeks, three weeks, or fourweeks after the pharmaceutically-acceptable formulation is administeredto the subject.

In certain embodiments, these pharmaceutical compositions are suitablefor topical or oral administration to a subject. In other embodiments,as described in detail below, the pharmaceutical compositions of thepresent invention may be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastes;(2) parenteral administration, for example, by subcutaneous,intramuscular or intravenous injection as, for example, a sterilesolution or suspension; (3) topical application, for example, as acream, ointment or spray applied to the skin; (4) intravaginally orintrarectally, for example, as a pessary, cream or foam; or (5) aerosol,for example, as an aqueous aerosol, liposomal preparation or solidparticles containing the compound.

The phrase “pharmaceutically acceptable” refers to those compound of theinventions of the present invention, compositions containing suchcompounds, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” includespharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier is “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions containing a compound of the invention(s) include thosesuitable for oral, nasal, topical (including buccal and sublingual),rectal, vaginal, aerosol and/or parenteral administration. Thecompositions may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. The amountof active ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the host beingtreated, the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, out of one hundred percent,this amount will range from about 1 percent to about ninety-nine percentof active ingredient, preferably from about 5 percent to about 70percent, more preferably from about 10 percent to about 30 percent.

Methods of preparing these compositions include the step of bringinginto association a compound of the invention(s) with the carrier and,optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation a compound of the invention with liquid carriers, or finelydivided solid carriers, or both, and then, if necessary, shaping theproduct.

Compositions of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of theinvention(s) as an active ingredient. A compound may also beadministered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compound of theinvention(s) include pharmaceutically-acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, the oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compound of the invention(s) maycontain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Pharmaceutical compositions of the invention for rectal or vaginaladministration may be presented as a suppository, which may be preparedby mixing one or more compound of the invention(s) with one or moresuitable nonirritating excipients or carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the rectum or vaginal cavity and releasethe active agent.

Compositions of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof the invention(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound ofthe invention(s) may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition tocompound of the invention(s) of the present invention, excipients, suchas animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of theinvention(s), excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The compound of the invention(s) can be alternatively administered byaerosol. This is accomplished by preparing an aqueous aerosol, liposomalpreparation or solid particles containing the compound. A nonaqueous(e.g., fluorocarbon propellant) suspension could be used. Sonicnebulizers are preferred because they minimize exposing the agent toshear, which can result in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically-acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the invention(s) to the body. Such dosageforms can be made by dissolving or dispersing the agent in the propermedium. Absorption enhancers can also be used to increase the flux ofthe active ingredient across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe active ingredient in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of the invention.

Pharmaceutical compositions of the invention suitable for parenteraladministration comprise one or more compound of the invention(s) incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers, which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofcompound of the invention(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compound of the invention(s) are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically-acceptable carrier.

Regardless of the route of administration selected, the compound of theinvention(s), which may be used in a suitable hydrated form, and/or thepharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels and time course of administration of the activeingredients in the pharmaceutical compositions of the invention may bevaried so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient. An exemplary dose range is from 0.1 to 10 mg per day.

A preferred dose of the compound of the invention for the presentinvention is the maximum that a patient can tolerate and not developserious side effects. Preferably, the compound of the invention of thepresent invention is administered at a concentration of about 0.001 mgto about 100 mg per kilogram of body weight, about 0.001-about 10 mg/kgor about 0.001 mg-about 100 mg/kg of body weight. Ranges intermediate tothe above-recited values are also intended to be part of the invention.

6. Screening Methods and Systems

In another aspect, the invention provides a machine readable storagemedium which comprises the structural coordinates of either one or bothof the binding pockets identified herein, or similarly shaped,homologous binding pockets. Such storage medium encoded with these dataare capable of displaying a three-dimensional graphical representationof a molecule or molecular complex which comprises such binding pocketson a computer screen or similar viewing device.

The invention also provides methods for designing, evaluating andidentifying compounds which bind to the aforementioned binding pockets.Thus, the computer produces a three-dimensional graphical structure of amolecule or a molecular complex which comprises a binding pocket.

In another embodiment, the invention provides a computer for producing athree-dimensional representation of a molecule or molecular complexdefined by structure coordinates of 5-HT2C or domains thereof, or athree-dimensional representation of a homologue of said molecule ormolecular complex, wherein said homologue comprises a binding pocketthat has a root mean square deviation from the backbone atoms of saidamino acids of not more than 2.0 (more preferably not more than 1.5)angstroms

In exemplary embodiments, the computer or computer system can includecomponents which are conventional in the art, e.g., as disclosed in U.S.Pat. Nos. 5,978,740 and/or 6,183,121 (incorporated herein by reference).For example, a computer system can includes a computer comprising acentral processing unit (“CPU”), a working memory (which may be, e.g.,RAM (random-access memory) or “core” memory), a mass storage memory(such as one or more disk drives or CD-ROM drives), one or morecathode-ray tube (CRT) or liquid crystal display (LCD) displayterminals, one or more keyboards, one or more input lines, and one ormore output lines, all of which are interconnected by a conventionalsystem bus.

Machine-readable data of this invention may be inputted to the computervia the use of a modem or modems connected by a data line. Alternativelyor additionally, the input hardware may include CD-ROM drives, diskdrives or flash memory. In conjunction with a display terminal, akeyboard may also be used as an input device.

Output hardware coupled to the computer by output lines may similarly beimplemented by conventional devices. By way of example, output hardwaremay include a CRT or LCD display terminal for displaying a graphicalrepresentation of a binding pocket of this invention using a programsuch as QUANTA or PYMOL. Output hardware might also include a printer,or a disk drive to store system output for later use.

In operation, the CPU coordinates the use of the various input andoutput devices, coordinates data accesses from the mass storage andaccesses to and from working memory, and determines the sequence of dataprocessing steps. A number of programs may be used to process themachine-readable data of this invention, includingcommercially-available software.

A magnetic storage medium for storing machine-readable data according tothe invention can be conventional. A magnetic data storage medium can beencoded with a machine-readable data that can be carried out by a systemsuch as the computer system described above. The medium can be aconventional floppy diskette or hard disk, having a suitable substratewhich may be conventional, and a suitable coating, which may also beconventional, on one or both sides, containing magnetic domains whosepolarity or orientation can be altered magnetically. The medium may alsohave an opening (not shown) for receiving the spindle of a disk drive orother data storage device.

The magnetic domains of the medium are polarized or oriented so as toencode in manner which may be conventional, machine readable data suchas that described herein, for execution by a system such as the computersystem described herein.

An optically-readable data storage medium also can be encoded withmachine-readable data, or a set of instructions, which can be carriedout by a computer system. The medium can be a conventional compact diskread only memory (CD-ROM) or a rewritable medium such as amagneto-optical disk which is optically readable and magneto-opticallywritable.

In the case of CD-ROM, as is well known, a disk coating is reflectiveand is impressed with a plurality of pits to encode the machine-readabledata. The arrangement of pits is read by reflecting laser light off thesurface of the coating. A protective coating, which preferably issubstantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, adata-recording coating has no pits, but has a plurality of magneticdomains whose polarity or orientation can be changed magnetically whenheated above a certain temperature, as by a laser. The orientation ofthe domains can be read by measuring the polarization of laser lightreflected from the coating. The arrangement of the domains encodes thedata as described above.

Structure data, when used in conjunction with a computer programmed withsoftware to translate those coordinates into the 3-dimensional structureof a molecule or molecular complex comprising a binding pocket may beused for a variety of purposes, such as drug discovery.

For example, the structure encoded by the data may be computationallyevaluated for its ability to associate with chemical entities. Chemicalentities that associate with a binding pocket of a 5-HT2C or specificdomains thereof, and are potential drug candidates. Alternatively, thestructure encoded by the data may be displayed in a graphicalthree-dimensional representation on a computer screen. This allowsvisual inspection of the structure, as well as visual inspection of thestructure's association with chemical entities.

Thus, according to another embodiment, the invention relates to a methodfor evaluating the potential of a chemical entity to associate with a) amolecule or molecular complex comprising a binding pocket of 5-HT2C orspecific domains thereof, or b) a homologue of said molecule ormolecular complex, wherein said homologue comprises a binding pocketthat has a root mean square deviation from the backbone atoms of saidamino acids of not more than 2.0 (more preferably 1.5) angstroms.

This method comprises the steps of:

i) employing computational means to perform a fitting operation betweenthe chemical entity and a binding pocket of the molecule or molecularcomplex; and

ii) analyzing the results of the fitting operation to quantify theassociation between the chemical entity and the binding pocket. The term“chemical entity”, as used herein, refers to chemical compounds,complexes of at least two chemical compounds, and fragments of suchcompounds or complexes.

The design of compounds that bind to 5-HT2C or specific domains thereofbinding pockets according to this invention generally involvesconsideration of several factors. First, the entity must be capable ofphysically and structurally associating with parts or all of the 5-HT2Cor specific domains thereof-related binding pockets. Non-covalentmolecular interactions important in this association include hydrogenbonding, van der Waals interactions, hydrophobic interactions andelectrostatic interactions. Second, the entity must be able to assume aconformation that allows it to associate with the 5-HT2C, or specificdomains thereof, binding pocket(s) directly. Although certain portionsof the entity will not directly participate in these associations, thoseportions of the entity may still influence the overall conformation ofthe molecule. This, in turn, may have a significant impact on potency.Such conformational requirements include the overall three-dimensionalstructure and orientation of the chemical entity in relation to all or aportion of the binding pocket, or the spacing between functional groupsof an entity comprising several chemical entities that directly interactwith the binding pocket or homologues thereof.

The potential binding effect of a chemical entity on a 5-HT2C orspecific domains thereof may be analyzed prior to its actual synthesisand testing by the use of computer modeling techniques. If thetheoretical structure of the given entity suggests insufficientinteraction and association between it and the target binding pocket,testing of the entity is obviated. However, if computer modelingindicates a strong interaction, the molecule may then be synthesized andtested for its ability to bind to a binding pocket. This may beachieved, e.g., by testing the ability of the molecule modulate 5-HT2C,or specific domains thereof, binding activity, e.g., using assaysdescribed herein or known in the art. In this manner, synthesis ofinoperative compounds may be avoided.

A potential binder of 5-HT2C or specific domains thereof (e.g., bindingpocket) may be computationally evaluated by means of a series of stepsin which chemical entities or fragments are screened and selected fortheir ability to associate with the 5-HT2C or specific domainsthereof-related binding pockets.

One skilled in the art may use one of several methods to screen chemicalentities or fragments for their ability to associate with a 5-HT2C orspecific domains thereof-related binding pocket. This process may beginby visual inspection of, for example, 5-HT2C or specific domainsthereof-related binding pocket on the computer screen based on the5-HT2C, or specific domains thereof structure coordinates describedherein, or other coordinates which define a similar shape generated fromthe machine-readable storage medium. Selected fragments or chemicalentities may then be positioned in a variety of orientations, or docked,within that binding pocket as defined supra. Docking may be accomplishedusing software such as Quanta and DOCK, followed by energy minimizationand molecular dynamics with standard molecular mechanics force fields,such as CHARMM and AMBER.

Specialized computer programs (e.g., as known in the art and/orcommercially available and/or as described herein) may also assist inthe process of selecting fragments or chemical entities.

Once suitable interacting/binding compound chemical entities orfragments have been selected, they can be assembled into a singlecompound or complex. Assembly may be preceded by visual inspection ofthe relationship of the fragments to each other on the three-dimensionalimage displayed on a computer screen in relation to the structurecoordinates of the target binding pocket.

Instead of proceeding to build an of a binding pocket in a step-wisefashion one fragment or chemical entity at a time as described above,interacting or other binding compounds may be designed as a whole or “denovo” using either an empty binding site or optionally including someportion(s) of a known interacting/binding compound(s). There are many denovo ligand design methods known in the art, some of which arecommercially available (e.g., LeapFrog, available from TriposAssociates, St. Louis, Mo.).

Other molecular modeling techniques may also be employed in accordancewith this invention [see, e.g., N. C. Cohen et al., “Molecular ModelingSoftware and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp.883-894 (1990); see also, M. A. Navia and M. A. Murcko, “The Use ofStructural Information in Drug Design”, Current Opinions in StructuralBiology, 2, pp. 202-210 (1992); L. M. Balbes et al., “A Perspective ofModern Methods in Computer-Aided Drug Design”, in Reviews inComputational Chemistry, Vol. 5, K. B. Lipkowitz and D. B. Boyd, Eds.,VCH, New York, pp. 337-380 (1994); see also, W. C. Guida, “Software ForStructure-Based Drug Design”, Curr. Opin. Struct. Biology, 4, pp.777-781 (1994)].

Once a compound has been designed or selected, the efficiency with whichthat entity may bind to a binding pocket may be tested and optimized bycomputational evaluation.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interactions. Examples of programsdesigned for such uses include: AMBER; QUANTA/CHARMM (Accelrys, Inc.,Madison, Wis.) and the like. These programs may be implemented, forinstance, using a commercially-available graphics workstation. Otherhardware systems and software packages will be known to those skilled inthe art.

Another technique involves the in silico screening of virtual librariesof compounds, e.g., as described herein. Many thousands of compounds canbe rapidly screened and the best virtual compounds can be selected forfurther screening (e.g., by synthesis and in vitro testing). Smallmolecule databases can be screened for chemical entities or compoundsthat can bind, in whole or in part, to a GPCR, 5-HT2c, or specificdomains thereof In this screening, the quality of fit of such entitiesto the binding site may be judged either by shape complementarity or byestimated interaction energy.

The compounds herein are also advantageous in that they possess a higherlevel of structural rigidity relative to previously reported 5-HT2active compounds. This advantage is useful for probing structuralinformation about GPCR targets (e.g., histamine, serotonin, muscarinic,etc.) and sub-families thereof. Such information is useful inelucidating binding pocket information, homology, sequence, etc. Thus,in aspects, the invention includes the use of the compounds delineatedherein as probes for elucidation of target structure information. Theuse includes methods for studying interaction and function of thecompounds herein with a PGCR target (both using “wet lab” experimentalassays, probes, crystallization techniques and protocols, proteinstudies, as well as in silico methods using representations of thecompounds).

Examples

The invention is further illustrated by the following examples which areintended to illustrate but not limit the scope of the invention.

Example 1 Database of Small Molecules

The NCI/DTP maintains a repository of approximately 240,000 samples(i.e., the plated compound set) which are non-proprietary and offered tothe research community for discovery and development of new agents forthe treatment of cancer, AIDS, or opportunistic infections afflictingsubjects with cancer or AIDS. The three-dimensional coordinates for theNCI/DTP plated compound set is obtained in the MDL SD format(http://www.chm.tu-dresden.de/edv/vamp65/REFERS/vr_(—)03d.htm) andconverted to the mol2 format by the DOCK utility program SDF2MOL2.Partial atomic charges, solvation energies and van der Waals parametersfor the ligands are calculated using SYBDB and added to the platedcompound set mol2 files.

Example 2 Database Screening to Identify Potential Small MoleculeInteracting/Binding Compounds.

In lieu of conducting high-throughput screening, a more rapid andeconomical structure-based approach combining molecular docking insilico with functional testing is used. A large chemical library ofcompounds with known three-dimensional structure is positioned in thestructural pocket selected by SPHGEN (UCSF) on the crystal structure ofa GPCR. This approach combines resources available through the NCI/DTP(atomic coordinates and small molecules) with improved molecular dockingand scoring algorithms imposed in DOCK5.1 (UCSF). 20,000 small moleculecompounds with drug-like characteristics (following the Lipinski rules)were docked into the 5-HT2c crystal structure in 100 differentorientations using DOCK5.1. The compounds with the highest scores arerequested for functional testing from the NCI/DTP.

The National Cancer Institute/Developmental Therapeutics Program(NCI/DTP) maintains a repository of approximately 220,000 samples (theplated compound set) that are nonproprietary and offered to theextramural research community free of charge. The three-dimensionalcoordinates for the NCI/DTP plated compound set was obtained in the MDLSD format and converted to the mol2 format by the DOCK utility programSDF2MOL2. Partial atomic charges, solvation energies, and van der Waalsparameters for the ligands were calculated using SYBDB and added to theplated compound set mol2 file.

In Silico Molecular Docking of Potential Small MoleculeInteracting/Binding Compounds.

All docking calculations are performed with the DOCK, v5.1.0. Thegeneral features of DOCK include rigid orienting of ligands to receptorspheres, AMBER energy scoring, GB/SA solvation scoring, contact scoring,internal nonbonded energy scoring, ligand flexibility, and both rigidand torsional simplex minimization. Unlike previously distributedversions, this release incorporates automated matching, internal energy(used in flexible docking), scoring function hierarchy, and newminimizer termination criteria. The coordinates for the molecular modelof the 5-HT2c domain are used in the molecular docking calculations. Toprepare the site for docking, all water molecules are removed.Protonation of receptor residues is performed with Sybyl (Tripos, St.Louis, Mo.). The structure is explored using sets of spheres to describepotential binding pockets. The number of orientations per molecule is100. Intermolecular AMBER energy scoring (vdw+columbic), contactscoring, and bump filtering are implemented in DOCK5.1.0. SETOR andGRASP are used to generate molecular graphic images.

As shown herein, representative compounds herein have GPCR modulatingactivity.

Example 3 Chemicals

(1R,3S)-(−)-Trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene(trans-PAT, FIG. 1) was synthesized by modification of a procedurepreviously reported (Wyrick et al., 1993). Briefly,(E)-1,4-diphenylbut-1-en-3-one was cyclized to the correspondingtetralone intermediate using polyphosphoric acid in toluene under refluxconditions (18 h) and the product was purified by flash columnchromatography. The tetralone was reduced with sodium borohydride to amixture of (±)-cis- and (±)-trans-tetralols that could be separated byrecrystallization. The (±)-cis-tetralol was stirred withp-toluenesulfonyl chloride in pyridine for 2 days at room temp to obtainthe corresponding tosylate intermediate. Stirring the tosylate withsodium azide in N,N-dimethylformamide for 2 days at room temp yieldedthe (±)-trans-azido derivative, which was reduced by catalytichydrogenation to the free amine. The (±)-trans-amine compound wasconverted to a diastereomeric salt using D-(−)-tartaric acid and thediastereomers were separated by fractional recrystallization. The pure(1R,3S)-(−)-trans-amine was dimethylated using formic acid/formaldehydeand purified by flash column chromatography to obtain the pure(1R,3S)-(−)-trans-PAT product.

[³H]-Ketanserin (specific activity 72.2 Ci/mmol) andmyo-[2-³H(N)]-Inositol (specific activity 18.5 Ci/mmol) were purchasedfrom Perkin-Elmer Life Science (Boston, Mass.) and[N⁶-methyl-³H]-mesulergine (specific activity 72.0 Ci/mmol) fromAmersham Biosciences (GE healthcare, Piscataway, N.J.). Other compoundswere obtained in highest purity from Sigma-Aldrich (St. Louis, Mo.).

Clonal Cell Culture and Transfection

All cell lines were maintained by following ATCC suggestion, ChineseHamster Ovary cells (CHO-K1, ATCC CCL-61) in Ham's F-12 mediumsupplemented with 10% fetal bovine serum, 1% sodium bicarbonate(Mediatech 25-035-CI), 10 IU/ml Penicillin and 10 ug/ml Streptomycin,and human embryonic kidney (HEK) 293 in minimum essential medium (Eagle)(MEM) with 2 mM L-glutamine adjusted to contain 1.5 g/L sodiumbicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodiumpyruvate (90%) with 10% fetal bovine serum, 10 IU/ml Penicillin and 10ug/ml Streptomycin. Cells were grown at 37° C. in a humidified incubatorwith 5% CO2. The cDNAs encoding the human 5-HT_(2A), 5-HT_(2B), and5-HT_(2C) receptors (wild type) were purchased from UMR (Rolla, Mo.) fortransient transfection of the clonal cells. For radioreceptor bindingassays, 5-HT_(2A), 5-HT_(2B), and 5-HT_(2C) receptor membranes wereprepared from transfected CHO-K1 cells. For functional assays measuringactivity of PLC/IP formation, transfected CHO-K1 cells were used for5-HT_(2A) and 5-HT_(2C) receptors. For 5HT_(2B) receptors, however, morerobust and consistent results for the PLC/IP assay were obtained usingtransfected HEK cells (Setola et al., 2005). Twenty-four hours beforetransfection, cells were seeded at 40% confluence in 100 mm dishes forradioreceptor binding assays or at 10⁵ cells per well in 12-well platesfor functional assays. CHO-K1 cells were transiently transfected with 12μg of plasmid and 32 μl of lipofectamine (Invitrogen) per 100 mm dishfor radioreceptor binding assays, or, 0.8 μg plasmid and 4.0 μl oflipofectamine per well for functional assays. For 5-HT_(2B) functionalassays using HEK cells, 24 μg plasmid DNA was mixed with 60 μl ofLipofectamine 2000 (Invitrogen) to transfect 1-2×10⁶ cells in a 10-cmplate. Cells were allowed to express transfected receptors for another24 hrs (Herrick, 1997).

Radioreceptor Assays

Radioreceptor saturation and competition binding assays were performedusing membrane homogenates, similar to our methods reported previouslyfor the phylogenetically closely related histamine H₁ GPCR (Booth, 2002;Moniri et al., 2004). [³H]-Ketanserin was used to radiolabel 5-HT_(2A)receptors and [³H]-mesulergine for 5-HT_(2B) and 5-HT_(2C) receptors.Briefly, forty-eight hours following CHO cell transfection, cells wereharvested and homogenized in 50 mM Tris-HCl containing 0.1% ascorbicacid and 4.0 mM CaCl₂ at pH 7.4 (assay buffer). The homogenate wascentrifuged at 35,000 g for 25 min and the resulting membrane pellet wasre-suspended in assay buffer. Protein concentration was determined bythe method of Lowry et al. (Lowry, 1951). For saturation binding assays,membrane suspension containing 100 μg protein was incubated with 0.1-5.0nM [³H]-ketanserin (5-HT_(2A) receptors) or 0.1-20 nM [³H]-mesulergine(5-HT_(2B) and 5-HT_(2C) receptors) in a total assay buffer volume of250 μl. Non-specific binding was determined in the presence of 10 μMmethysergide (5-HT_(2A) receptors) or 1.0 μM mianserin (5-HT_(2B) and5-HT_(2C) receptors). Competition binding assays were conductedsimilarly with 1.0 nM [³H]-ketanserin or [³H]-mesulergine. Incubation ofradioreceptor binding assay mixtures was for 1.0 h at 37° C., withtermination by rapid filtration through Whatman GF/B filters using a96-well cell harvester (Tomtec, Hamden, Conn.). The membrane-bound[³H]-radioligand retained on the filter discs was quantified by liquidscintillation spectrometry. Data were analyzed by nonlinear regressionusing the sigmoidal curve-fitting algorithms in Prism 4.03 (GraphPadSoftware Inc., San Diego, Calif.). Ligand affinity is expressed as anapproximation of K_(i) values by conversion of the IC₅₀ data to K_(0.5)values using the equation K_(0.5)=IC₅₀/1+L/K_(D), where L is theconcentration of radioligand having affinity K_(D) (Cheng, 1973). Eachexperimental condition was performed in triplicate and each experimentwas performed a minimum of three times to determine S.E.M.

Assay for Activation of PLC and [³H]-IP Formation

Functional activation of PLC was measured as [³H]-IP formation in CHOcells transiently expressing serotonin 5-HT_(2C) receptors or HEK cellstransiently expressing serotonin 5-HT_(2A) or 5-HT_(2B) receptors, aspreviously reported (Moniri et al., 2004). Briefly, thirty-two hoursfollowing transfection, cells in inositol-free Dulbecco's modifiedEagle's medium (DMEM) were incubated for twelve hours with 1.0 μCi/mlmyo-[2-³H]-inositol, the radiolabeled precursor of the PLC-β substratephosphatidylinositol. Cells then were washed and incubated in DMEMcontaining 10 mM lithium chloride, 10 μM pargyline (with addition of 5%dialyzed fetal bovine serum for HEK cells), and, various concentrationsof test ligand for 45-60 min at 37° C. After aspiration of media, wellswere lysed by incubation with 50 mM formic acid (15-60 min). Formic acidwas neutralized with ammonium hydroxide and contents from each well wereadded to individual AG1-X8 200-400 formate resin anion exchange columns.Ammonium formate/formic acid (1.2 M/0.1 M) was used to elute [³H]-IPdirectly into scintillation vials for counting of tritium by liquidscintillation spectrometry. Resulting data were analyzed using thenonlinear regression algorithms in Prism 4.03 and are expressed as meanpercentage of control [³H]-IP formation, with potency expressed asconcentration required to stimulate (EC₅₀) or inhibit (IC₅₀) maximalbasal (constitutive) [³H]-IP formation by 50%±S.E.M. (n≧3).

Measurement of [³H]-IP Formation in CHO-K1 and HEK Cells

Functional activation of PLC was measured as [³H]-IP formation in CHOcells transiently expressing 5-HT_(2A) or 5-HT_(2C) receptors and HEKcells transiently expressing 5HT_(2B) receptors, as previously reportedby our lab (Booth, 2002; Moniri et al., 2004). Briefly, thirty-two hoursfollowing transfection, cells in inositol-free Dulbecco's modifiedEagle's medium (DMEM) were labeled with 1 μCi/ml myo-[2-³H]-inositol, aprecursor of the PLC-β substrate phosphatidylinositol. Cells then werewashed and incubated in DMEM containing 25 mM Hepes (pH 7.4), 10 mMLiCl, 10 μM pargyline (with addition of 5% dialyzed FBS for HEK cells),and various concentrations of test ligand for 45-60 min at 37° C. Afteraspiration of media, wells were placed on ice and lysed by incubationwith 50 mM formic acid (15-60 min). Formic acid was neutralized withammonium hydroxide and all contents from each well were added toindividual AG1-X8 200-400 formate resin anion exchange columns. Ammoniumformate/formic acid (1.2 M/0.1 M) was used to elute [³H]-IP directlyinto scintillation vials for counting of tritium by liquid scintillationspectrometry. Resulting data were analyzed using the nonlinearregression algorithms in Prism 4.03 and are expressed as mean percentageof control [³H]-IP formation, with potency expressed as concentrationrequired to produce 50% maximal [³H]-IP formation (EC₅₀)±S.E.M (n≧3).

Example 4 Radioreceptor Assays

Radioligand saturation binding analysis of 5HT-subtype receptors: Therewas no measurable specific radioligand binding using membranes preparedfrom null-transfected CHO and HEK cells. Using membranes prepared fromCHO cells transiently transfected with 5-HT_(2A), 5-HT_(2B), or5-HT_(2C) cDNA, however, saturable specific radioligand bindingoccurs—representative binding curves for [³H]-ketanserin labeled5HT_(2A) receptors and [³H]-mesulergine labeled 5-HT_(2B) receptors and5-HT_(2C) receptors are shown in FIGS. 2A-C. [³H]-Ketanserin binds to anapparent single population of 5HT_(2A) receptors (B_(max)=1.73±0.11pmol/mg protein) with high affinity (K_(D)=0.80±0.03 nM). Similarly,[³H]-mesulergine labels a single population of 5HT_(2B) receptors withB_(max)=1.13±0.39 pmol/mg protein and K_(D)=5.19±0.36 nM.[³H]-mesulergine also labels an apparent single population of 5HT_(2C)receptors (B_(max)=8.37±0.15 pmol/mg prot) with high affinity(K_(D)=0.88±0.03 nM).

Example 5

Competition binding analysis to determine (−)-trans-PAT 5HT₂-subtypeReceptor affinity. Representative 5-HT_(2A), 5-HT_(2B), and 5-HT_(2C)radioligand displacement curves for (−)-trans-PAT are shown in FIG. 3.Curves are sigmoidal in shape and span 3-4 log ligand concentrationunits to achieve complete radioligand displacement, characteristic ofcompetitive displacement of ˜K_(D) radioligand concentration from asingle population of GPCRs. The K_(i)±SEM values for (−)-trans-PAT at5HT_(2A), 5-HT_(2B), and 5-HT_(2C) receptors are 410±38, 130±28, and37.6±3.02 nM (respectively), with corresponding n_(H) values of 1.1±0.1,1.1±0.1, and 0.9±0.1.

Example 6 Functional Assays

Assessment of (−)-trans-PAT agonist activity at 5HT₂-subtype receptors:The 5HT₂ GPCR family is constitutively active when expressed in CHO andHEK cells, thus, functional activity here is reported relative to basalactivity of PLC/[³H]-IP formation in FIG. 4. In lysates ofnull-transfected CHO and HEK cells, no increase in basal activity ofPLC/[³H]-IP formation was detected after incubation with up to 10 μM5-HT for 45 min. In CHO cells transiently transfected with human5-HT_(2C) cDNA, however, 5-HT produces a concentration-dependentincrease in basal activity of PLC/[³H]-IP formation, with EC₅₀=6.30±0.55nM (n_(H)=1.3±0.2) and E_(max)˜0.1 μM (˜475% basal control activity), asshown in the FIG. 4 inset. Relative to the endogenous agonist,(−)-trans-PAT is a full-efficacy 5HT_(2C) agonist that produces aconcentration-dependent increase in basal activity of PLC/[³H]-IPformation, with EC₅₀=21.4±2.22 nM (n_(H)=0.66±0.11) and E_(max)˜10 μM(˜475% basal control activity) (FIG. 4). In CHO cells transientlytransfected with human 5HT_(2A) cDNA, however, (−)-trans-PAT did notstimulate PLC/[³H]-IP formation at concentrations up to 10 μM; forcomparison, 5HT EC₅₀=30±2 nM, E_(max)˜1.0 μM (˜300% basal controlactivity) (data not shown). Likewise, in HEK cells transientlytransfected with human 5HT_(2B) cDNA, (−)-trans-PAT did not stimulatePLC/[³H]-IP formation at concentrations up to 30 μM; for comparison, 5HTEC₅₀=19.7±9.21 nM, E_(max)˜1.0 μM (˜900% basal control activity (datanot shown).

Example 7

Assessment of (−)-trans-PAT antagonist activity at 5HT₂-subtypereceptors: Given that (−)-trans-PAT binds with moderate affinity to5HT_(2A) and 5HT_(2B) receptors (FIG. 3) but does not activate these5-HT2 receptor subtypes (FIG. 4), the ability of (−)-trans-PAT to act asa 5HT_(2A) and 5HT_(2B) receptor antagonist regarding 5-HT-mediatedstimulation of PLC/[³H]-IP formation was assessed and results are shownin FIG. 5. In CHO cells expressing human 5HT_(2A) receptors, 5-HT (1.0μM) stimulated PLC/[³H]-IP formation (˜250% basal control) and thiseffect was fully blocked by (−)-trans-PAT (10 μM) (FIG. 5A). In HEKcells expressing human 5HT_(2B) receptors, 5-HT (0.01 μM) stimulatedPLC/[³H]-IP formation (˜350% basal control) and this effect was fullyblocked by (−)-trans-PAT (3.0 μM) (FIG. 5B).

Example 8 Discussion

The data reported here indicate that relative to the endogenous agonistserotonin, (−)-trans-PAT is a stereoselective full-efficacy agonist athuman serotonin 5-HT_(2C) receptors. The selectivity of (−)-trans-PATfor activation of 5-HT_(2C) receptors versus 5-HT_(2A) and 5-HT_(2B)receptors is unequivocal in light of its competitive antagonism ofserotonin activation of 5-HT_(2A) and 5-HT_(2B) receptors, and, itsinherent inverse agonist functional activity at 5-HT_(2A) and 5-HT_(2B)receptors. The unique multifunctional activity of (−)-trans-PAT atserotonin 5-HT₂-type receptors is promising with regard to developmentof novel 5-HT_(2C) receptor-based pharmacotherapy. Results indicate that(−)-trans-PAT demonstrates full-efficacy for activation of human5HT_(2C) receptors as well as inverse agonist and/or antagonist activityat 5HT_(2A) and 5HT_(2B) receptors. These results have promisingimplications for development of novel 5HT_(2C)-based pharmacotherapy.For example, activation of brain 5HT_(2C) receptors is well establishedto be effective pharmacotherapy for obesity (Tecott et al., 1995;Vickers et al., 1999; 2001; Heisler et al., 2002). Meanwhile, therecurrently is no effective pharmacotherapy for cocaine addiction. It isthought, however, that disorders involving over-eating and drugself-administration are members of the same group of compulsivebehavioral disorders directed toward different objects, food and drugs(Simansky, 2005). Accordingly, a role for brain 5HT_(2C) receptoractivation in pharmacotherapy of both obesity and cocaine addictionappears logical. In fact, the balance of studies using reliable 5HT₂subtype-selective antagonists suggest that the reinforcing effects ofcocaine are reduced by 5HT_(2C) activation, and, discriminative stimulusand reinstating effects of cocaine are sensitive to attenuation by5HT_(2C) activation as well as by 5HT_(2A) antagonism (Bubar andCunningham, 2006).

In contrast, 5HT_(2A) receptor activation is closely associated withpsychomimetic activity (Nichols, 2004), as perhaps best demonstrated bythe inverse observation that drugs with 5HT_(2A) antagonist activity areeffective to treat psychosis and related neuropsychiatric disorders(Baldessarini and Tarazi, 2006). Thus, the possibility of 5HT_(2A)receptor activation concomitant with 5HT_(2C) activation for drugs thatshow only modest 15-fold selectivity in vitro, such as lorcaserin(Jensen, 2006; Smith et al., 2006), should be carefully considered inview of the sometimes subtle and complex nature of psychiatricdisturbances. Perhaps, the life-threatening cardiac valvulopathy andpulmonary hypertension associated with fenfluramine has led to a higherthreshold for selectivity (e.g., 100-fold, in vitro) regardingdevelopment of drugs that activate both 5HT_(2C) and 5HT_(2B) receptors.Nevertheless, clinical use of a diet drug by perhaps 30-50 millionpeople (Nilsson, 2006; Smith et al., 2006), often chronically, oftenunsupervised, could lead to problems if the drug demonstrates anyactivation of 5HT_(2A) and/or 5HT_(2B) receptors. We have made an extraeffort to ensure that any lead compounds we put forward for drugdevelopment demonstrate unequivocal 5HT_(2B) antagonism by switching toHEK cells because this clonal cell line gives more robust andreproducible functional results compared to CHO cells for the 5HT_(2B)receptor. Thus, a 5HT_(2C) agonist such as (−)-trans-PAT, thatdemonstrates unequivocal 5HT_(2A) and 5HT_(2B) inverse agonism and/orantagonism, and is a small lipophilic molecule that penetrates mammalianbrain after peripheral administration, appears to be well-suited forconsideration as novel pharmacotherapy for obesity, cocaine addiction,psychosis, anxiety, and, perhaps other neuropsychiatric disorders. Inthis regard, the unequivocal 5-HT_(2A) inverse agonist/antagonistactivity demonstrated by (−)-trans-PAT (see, FIGS. 17, 18) suggestspsychiatric side-effects linked to 5-HT_(2A) receptor activation likelywould not be an issue for this compound.

Example 9

1b. Activity of PAT Analogs at GPCRs

Affinity of PAT analogs in Tables 1 for the histamine H₁ GPCR is knownand Ki range is 0.5-5,000 nM (Ghoneim et al., 2006; Booth et al., 2002;Bucholtz et al., 1999; each incorporated by reference therein). Asmentioned, there is virtually no molecular information published aboutligand-5HT_(2C) receptor binding site interactions, but, our preliminarydata indicate there are differences in comparison to the H₁ receptor.There is information about the H₁ binding site from computationalchemistry (QSAR) and receptor modeling studies (e.g., Ghoneim et al.,2006; Jongejan and Leurs, 2005; Jongejan et al., 2005) that facilitatesprediction of ligand H₁ affinity. The relative lack of correspondingstudies for the 5HT_(2C) receptor, however, does not allow forprediction of which H₁-active ligands will have affinity at 5HT_(2C)receptors.

A number of the PAT analogs have been evaluated for H₁ functionalactivity. In view of the apparently subtle and largely unknown moleculardeterminants that govern ligand activation of a GPCR, it is perhaps notsurprising that most of the PATs are H₁ antagonists, but 1 and 6activate H₁-linked AC and PLC signaling, respectively (Moniri et al.,2004; Moniri and Booth, 2006). For both H₁ binding and function,stereochemistry significantly influences activity.

In addition to our H₁ and 5HT₂ results reported herein, several analogsin Table 1 were evaluated for affinity at a wide variety of CNSreceptors (PDSP, 2005; Novascreen, 1996). Our lead 5HT_(2C) agonistmolecule, (−)-trans-PAT (1), has very low (K_(i)>0.5 μM) or virtually no(K_(i)>5 μM) affinity for ˜35 radiolabeled CNS receptor systems,including, neurotransmitter (adrenergic α_(2A), α₁, β₁, β₂; cholinergic;GABA; glycine; histamine H₂, H₃, H4; serotonin 5HT_(1A), 5HT_(1B)),neuromodulator (adenosine, benzodiazepine, opiate, NMDA/PCP, sigma),neurotransporter (DAT, NET, SERT), ion channel (Ca⁺⁺, Cl⁻, K⁺), andsecond messenger systems (AC, PLC). The affinity (Ki) of racemic(±)-trans-PAT for 5HT_(5A), 5HT₆, and 5HT₇ receptors is about 100, 200,and 0.5 nM, respectively, and, affinity at adrenergic α_(2B) and α_(2C)receptors is 150 and 300 nM, respectively (PDSP, 2005).

1c. Radioligand Saturation Binding Experiments for Human 5HT_(2A),5HT_(2B), and 5HT_(2C) Receptors Transiently Expressed in CHO Cells(FIGS. 2-4).

Radioreceptor assays were set up in our lab for 5HT_(2A), 5HT_(2b), and5HT_(2C) receptors and saturation binding analysis was performed todetermine, in our hands, respective radioligand K_(D) and B_(max).Twenty-four hours before transfection, CHO-K cells were seeded at 40%confluence in 100 mm dishes for binding assays (or at 10⁵ cells per wellin 12-well plates for [³H]-IP assay, below). The cDNAs encoding thehuman 5-HT_(2A), 5-HT_(2B), or 5-HT_(2C) receptor (wild type) werepurchased from UMR resource center (Rolla, Mo.). Cells were transfectedwith 12 μg of plasmid and 32 μl lipofectamine (Invitrogen) per 100 mmdish or 0.8 μg of plasmid and 4.0 μl of lipofectamine (Invitrogen) perwell according to manufacturer protocols, our previous experience(Ghoneim et al, 2006; Moniri et al, 2004; Booth et al., 2002) and theliterature (Herrick-Davis et al, 1997). Receptors were radiolabeled withstandard 5HT₂ antagonist radioligands, using [³H]-ketanserin for5HT_(2A) receptors and [³H]-mesulergine for 5HT_(2B), and 5HT_(2C)receptors (Knight et al., 2004). Representative curves are in FIGS. 2-4and K_(D) and B_(max) summary in Table 3; values are consistent withliterature (Knight et al., 2004).

TABLE 3 K_(D) ± SEM B_(max) ± SEM (pmol/mg (nM) prot) 5HT_(2A) 0.80 ±0.03 1.73 ± 0.11 5HT_(2B) 5.20 ± 0.61 1.13 ± 0.67 5HT_(2C) 0.88 ± 0.038.37 ± 0.15

In view of unknown functional activity for most of our novel ligands,agonist radioligands were not used here because they label only asubpopulation of receptors in the “agonist-preferring” conformation(Knight et al., 2004; Sleight et al. 1996; Roth et al. 1998;Lopez-Gimenez et al. 2001; Quirk et al. 2001). Agonist-preferringconformation(s) for the 5HT₂ GPCR family is accounted for within theframework of the three-state model of GPCR activation, wherein GPCRsisomerize between inactive and constitutively active states (Kenakin,2001). A critical assumption of revised GPCR signaling theory is that aheterogeneity of active receptor conformations exists and that agonistligands differ in their ability to induce, stabilize, or select amongreceptor conformations. It follows that agonist ligand chemicalstructural parameters are among the most important determinants of GPCRconformation (Moniri et al., 2004). Currently there are no selective5HT_(2C) agonist radioligands available, thus, selective PAT 5HT_(2C)agonists with an N-alkyl moiety and Ki<5 nM will be considered forradiolabeling via our published procedures (Wyrick et al., 1992; 1994)

1d. Competition Binding Experiments:

Affinity of Trans-(−)-PAT Stereoisomers at 5HT_(2c) Receptors (FIG. 5,Table 4).

The 5HT_(2C) receptor affinity of (−)-trans-PAT and its stereoisomers,(+)-trans-PAT, (−)-cis-PAT, and (+)-cis-PAT, was assessed in competitiveradioligand displacement assays using ˜K_(D) concentration ofradioligand (as determined above). Curves (FIG. 5) are sigmoidal andspan 3-4 log concentration units to achieve complete radioliganddisplacement, characteristic of competitive displacement of ˜K_(D)radioligand concentration. The Hill coefficient (n_(H)) for the slope ofthe competitive displacement curve for (−)-trans-PAT is 0.9,characteristic of agonist ligand binding at a GPCR, according to theternary complex model with limiting availability of G protein, and,subpopulation(s) of receptor in an agonist-preferring conformation(s).The n_(H) values for the other PAT stereoisomers range 0.8-1.0(antagonists theoretically should have n_(H)=1). Except for(−)-trans-PAT, none of the other PAT stereoisomers activate 5HT_(2C)receptors at concentrations up to 10 μM (i.e., at least 10-times Ki)—seeAim 2 results. The stereoselectivity of 5HT_(2C) receptors for PATisomers has significant applications to delineate the 3D structure ofthe 5HT_(2C) active site and molecular determinants for receptoractivation.

TABLE 4 PAT Stereoisomer 5HT_(2C) Ki ± SEM n_(H) (1R,3S)-(−)-trans-PAT 37.6 ± 3.0 nM 0.9 (+/−)-trans-PAT 75.0 nM ± 2.2 nM 0.7 (not shown inFIG. 5 for clarity) (1S,3R)-(+)-trans-PAT 1270 ± 84.8 nM 1.0(1S,3S)-(−)-cis-PAT  433 ± 4.8 nM 0.8 (1R,3R)-(+)-cis-PAT  975 ± 7.8 nM0.81e. Affinity of Trans-(−)-PAT Stereoisomers at 5HT_(2A) Receptors (FIG.6).

Concentration-response curves for radioligand displacement by(−)-trans-PAT and its stereoisomers at 5HT_(2A) receptors is shown inFIG. 6. The 5HT_(2A) affinity of (−)-trans-PAT (Ki˜400 nM) is 10-foldlower than at 5HT_(2C) receptors. Table 5 summarized Ki and n_(H) valuesfor all 4 PAT stereoisomers. The rank order for affinity of PATstereoisomers at 5HT_(2A) receptors is different than at 5HT_(2C)receptors; rank order at 5HT_(2A) also differs from histamine H₁receptors. The n_(H) value for (−)-trans-PAT at 5HT_(2A) is 0.9,however, it is a 5HT_(2A) antagonist (FIGS. 10,11). The functional assayis not sensitive enough to detect inverse agonism, but, such activity islikely. Functional assessment of other PAT stereoisomers at 5HT_(2A)(n_(H)=0.9-1.0) is not complete.

1f. Affinity of Trans-(−)-PAT Stereoisomers at 5HT_(2B) Receptors (FIG.7).

Concentration-response curves for radio-ligand displacement by(−)-trans-PAT and its stereoisomers at 5HT_(2B) receptors is shown inFIG. 7. The 5HT_(2B) affinity of (−)-trans-PAT (Ki˜1 μM) is 20-foldlower than at 5HT_(2C) receptors. The n_(H) value for (−)-trans-PAT at5HT_(2A) is 1.0, consistent with its 5HT_(2B) antagonist activity (FIGS.10,12). Ki and n_(H) values for all 4 PAT stereoisomers are summarizedin Table 5. Rank order of PAT stereoisomer binding at 5HT_(2B) receptorsis different and overall much lower than at 5HT_(2A) and 5HT_(2C)receptors. Thus, although amino acid sequence is very similar formembers of the 5HT₂ family, the 3D arrangement of amino acids that formthe PAT ligand binding site appears to be different (especially for the5HT_(2B) receptor). Thus the PAT stereochemical scaffold can be used asa template for molecular modeling (structural) studies and optimized toprovide drugs with selective actions at 5HT₂ subtypes.

TABLE 5 PAT Stereoisomer 5HT_(2A) Ki ± SEM n_(H) 5HT_(2B) Ki ± SEM n_(H)5HT_(2C) Ki ± SEM n_(H) (1R,3S)-(−)-trans-PAT 407.3 ± 38.4 nM 0.91,168.8 ± 6.3 nM 1.0 37.6 ± 3.0 nM  0.93 (1S,3R)-(+)-trans-PAT 520.1 ±0.29 nM 1.0 ~2500 nM 1.0 1270 ± 84.8 nM  1.0 (1S,3S)-(−)-cis-PAT 1452.4± 0.23 nM  1.0 >5000 nM 433 ± 4.8 nM 0.80 (1R,3R)-(+)-cis-PAT 776.8 ±0.20 nM 0.9 >5000 nM 975 ± 7.8 nM 0.76

Example 10 In Vitro Characterization of PAT Functional Activity at 5HT₂Receptor Subtypes.

2a. (−)-Trans-PAT is a Stereospecific Full Efficacy Agonist at 5HT_(2C)Receptors (FIGS. 9-10).

The 5HT₂ GPCRs are constitutively active and predominantly activateGα_(q) protein to stimulate phospholipase (PL) C and inositol phosphates(IP) formation in mammalian tissues (Raymond et al., 2001). FIG. 9 showsthat in CHO-5HT_(2C) cells, (−)-trans-PAT is a 5HT_(2C) agonist(EC₅₀=21.4±2.22 nM, n_(H)=0.66) with full efficacy relative to 5HT(EC₅₀=6.30±0.55 nM, n_(H)=1.3) for stimulation of PLC/[³H]-IP formation.FIG. 9 also shows that the APT analog Cl-6APT (5HT_(2C) Ki˜300 nM, FIG.8), also is a full efficacy 5HT_(2C) agonist, but, it has very lowpotency (EC₅₀=4,630±312 nM; n_(H)=0.63) in comparison to (−)-trans-PATand 5HT.

The results for (−)-trans-PAT vs. Cl-6APT suggest that the position ofthe pendant phenyl ring is important, likely, for π-π stackinginteractions with 5HT_(2C) aromatic amino acids involved in receptorbinding and/or activation. Mutational analysis of the 5-HT_(2A) receptorsuggests important ligand-receptor π-π binding interactions occur inTMDs 5 & 6. (Shapiro et al., 2000)—this information helps to guidefuture 5HT_(2C) mutagenesis and molecular modeling studies.

(+)-Trans-, (−)-cis-, and (+)-cis-PAT also were evaluated for ability toactivate 5HT_(2C) receptors (PLC/[³H]-IP formation). FIG. 10 shows thatat 1.0 μM and 10 μM (i.e., at least 10-times Ki), (+)-trans-, (−)-cis-,and (+)-cis-PAT are not 5HT_(2C) agonists. Thus, the (−)-trans-PATfunctional effect is stereo-specific, consistent with the >30-fold rangein binding affinity between the stereoisomers at 5HT_(2C) receptors.

Interestingly, the potency of (−)-trans-PAT to activate 5HT_(2C)receptors (EC₅₀˜20 nM) is about 2-fold higher than its 5HT_(2C) affinitywhen measured using an antagonist radioligand (Ki˜40 nM, FIG. 5). Inthis regard it is noted that antagonist radioligands do not distinguishthe “agonist-preferring” conformation of the receptor (Roth et al. 1998;Lopez-Gimenez et al. 2001; Quirk et al. 2001). In fact, the Ki of(−)-trans-PAT for the agonist-preferring conformation of the 5HT_(2C)receptor may be closer to 20 nM rather than 40 nM. Studies will beconducted using the standard (but nonselective) 5HT₂ agonistradioligand, [³H]-2,5-dimethoxy-4-iodoamphetamine to confirm this.

2b. (−)-Trans-PAT Does Not Activate 5HT_(2A) or 5HT_(2B) Receptors (FIG.11).

As shown in FIG. 11, in CHO cells expressing human 5HT_(2A) or 5HT_(2B)receptors, (−)-trans-PAT did not activate PLC/IP formation, even at 10μM (˜25-times and 70-times 5HT_(2A) and 5HT_(2B) receptor Ki value,respectively). In parallel assays using CHO-5HT_(2C) cells, results weresimilar to those above. To the best of our knowledge, there is no otherligand reported that activates 5HT_(2C) receptors that does not alsoactivate 5HT_(2A) and/or 5HT_(2B) receptors. The results in FIG. 10suggest that (−)-trans-PAT is devoid of agonist activity at 5HT_(2A) and5HT_(2B) receptors. Thus, pharmacological studies to assess antagonistactivity were conducted, reported below.

2c. (−)-Trans-PAT is an Antagonist at 5HT_(2A) and 5HT_(2B) Receptors(FIGS. 12-13).

At 5HT_(2A) and 5HT_(2B) receptors, (−)-trans-PAT is an antagonist of5HT-mediated stimulation of PLC/IP formation (FIGS. 12, 13). At the5HT_(2B) receptor, we observed that (−)-trans-PAT at a concentration of1.0 μM (about Ki, Table 5) did not achieve full blockade (not shown),likely, due to the comparatively high affinity of 5HT for the 5HT_(2B)receptor (Ki˜1.0 nM). At a (−)-trans-PAT concentration of 10 μM,however, 5HT (1.0 μM) did not surmount the blockade, suggesting acompetitive antagonism. Experiments for full concentration-responsecurves to determine pA₂ values for (−)-trans-PAT at 5HT_(2A) and5HT_(2B) receptors will be undertaken in our lab.

Example 11 Synthesis of Meta-Substituted PAT Analogs and Separation ofEnantiomers (Schemes 1-3)

General synthetic methods: Details are described in our syntheticmedicinal chemistry publications (e.g., Ghoneim et al., 2006; Bucholtzet al., 1999; Wyrick et al., 1993; 1995). In vitro pharmacologicalstudies initially will use racemic cis and trans products. Racemic PATswith Ki<50 nM will be resolved to (+)- and (−)-enantiomers byderivatization to the diastereomeric salt followed by differentialcrystallization or synthesized de novo using a chiral reduction step(Scheme 3). Absolute configuration is assigned by single crystal X-raycrystallography or spectrophotometric methods (NMR, optical rotation) bycomparison to pure enantiomers already synthesized. Products (as HClsalts) characterized for purity using NMR, elemental analysis, massspectrometry, melting point and thin layer chromatography.

2. Synthesis of PAT Analogs with Fixed Phenyl Ring (FIG. 16)

The PAT pendant phenyl ring has a large degree of rotation flexibilityrelative to the tetrahydronapthalene scaffold, though it minimizes to alow energy orthogonal conformation. Analogous “pendant” phenyl ringsystems are present in a fixed configuration in high affinity 5HT_(2C)antagonist ligands such as mesulergine and ketanserin, but not in anyreported agonists. Fixing the PAT phenyl ring in an orthogonalconfiguration relative to the tetrahydronapthalene may enhance 5HT_(2C)affinity and provide information regarding PAT ligand-5HT_(2C) receptoraromatic (π-π) binding interactions. We propose synthesis of thebenzo[de]anthracene 1 and benzo[c]phenanthrene 2 rigid tetracyclicanalogs with a slightly curved conformation. In addition, we proposesynthesis of the spiroindane analog 3 wherein the phenyl ring is held attorsion angle 61°, similar to the X-ray crystal structure of(−)-trans-PAT (Wyrick et al., 1993); the related planar indanetetracyclic system in the tetrahydrofluoranthene analog 4 also will besynthesized.

i. 4,11-dihydro-5-N,N-dimethylamino-6H-benzo[de]anthracene (FIG. 16; 1):Anthrone is subjected to a Wittig reaction to give the acetate(Spinella, 1997). After reduction of the alkene and ester group usingNaBH₄ and PEG (Santaniello et al., 1981), the resultant alcohol is

converted to 9-bromoethyl-10-hydroanthracene with hydrobromic acid.Reaction of the bromide with sodium cyanide is followed by hydrolysis togive the acid and Friedel Crafts ring closure will produce the ketone5,11-dihydro-4-keto-6H-benzo[de]anthracene (Itoh et al., 1984) thatundergoes the same reactions as the corresponding PAT ketone 4 in Scheme3 to give 1 (FIG. 16).

ii. Cis & trans (+) &(−)-6-(dialkyl)amino-5,6,7,8,12b,6a-hexahydrobenzo[c]-phenanthrene (FIG.16; 2): The procedure of Laus (1984) will be used to prepare1-phenyl-2-tetralol which is oxidized to the tetralone. Wittig reactionand reduction will afford cis andtrans-1-phenyl-2-carbethoxymethylindane. Saponification will give thecorresponding acid which is cyclized to the ketone. Treatment of theketone with isoamylnitrite (Pandit and Huisman, 1966) affords the

corresponding α-carbonyl oxime. Reduction and methylation give 2.

iii. Cis & trans (+) &(−)-3′(dialkyl)-aminospiro[indan-2,1(2′H)-3,4-dihydro-naphthalene] (FIG.16; 3): The procedure of Majerus (1967) is used to preparebis(1-hydroxyindanyl) to2-oxospiro[indan-2,1(2′H)-3,4-dihydronaphthalene. This ketone is treatedwith isoamyl nitrite to afford the α-oxime followed by reduction underacidic conditions to the cis and trans primary amines which areN,N-dialkylated to give product 3.

iv. R- & S-2-Dimethylamino-10b,1,2,3,tetrahydrofluoranthene (FIG. 16;4): 1-(Fluoren-9-yl)-propanoate is prepared by formylation of fluoreneusing potassium methoxide and ethyl formate followed by Wittig reactionto afford the olefin which will be catalytically reduced and the estersaponified according to the procedure of Von and Wagner (1944). Thisacid will be ring closed with PPA to afford3-oxo-10b,1,2,3-tetrahydrofluoranthene. Reaction of this ketone withisoamyl nitrite to afford the α oxime followed by catalytic reduction tothe primary amine and N,N-alkylation to product 4 (FIG. 16).

Synthesis of New PAT Analogs with Changes to the C(1) Pendant PhenylSubstituent

Based on binding, function, 3D QSAR, and molecular modeling results inPreliminary Data, we hypothesize the (−)-trans-PAT C(1) pendant phenylmoiety is critical to providing full-efficacy 5HT_(2C) agonist activitywithout activation of 5HT_(2A) and 5HT_(2B) receptors. Testing PATpendant phenyl ring substitution and orientation will help to determineoptimal steric and electrostatic binding interactions with 5HT₂ activesite amino acids to obtain 5HT_(2C) agonists and/or 5HT_(2A)/5HT_(2B)antagonists with higher affinity, potency, and/or selectivity.

Scheme 1 shows the synthetic strategy for the new PATs in the Chartabove. In Step A, β-tetralone (1) is refluxed with benzene ruthenium(II) chloride dimer and the chiral ligand(R,R)-N-(2-amino-1,2-diphenylethyl)-p-toluenesulfonamide ((R,R)-NAPTS)to give the (R)-β-tetralol (2) (Mogi et al., 2004). In Step B, the(R)-β-tetralol (2) is converted to the tert-butyldimethylsilyl (TBDMS)derivative (3) (TBDMS protecting group to prevent bromination atadjacent benzylic position). In Step C, the TBDMS protected compound isbrominated with N-Bromosuccinimide (Agarwal et al., 1990) under refluxin anhydrous CCl₄ to give the brominated common intermediate (4),separated to cis and trans bromo compounds by flash columnchromatography. In Step D, each (cis and trans) brominated intermediate(4) is reacted with commercially available boronic acids (R₂B(OH)₂,R₂=a-p in Chart 1) under a Nickel-Catalyzed Suzuki

reaction (Gonzalez-Bobes et al) using NiI₂/trans-2-aminocyclohexanolwith sodium bis(trimethylsilyl)amide under reflux to make the variouscis and trans PAT analogs 50(a-p) shown in Table 2. In Step E, the TBDMSprotected PAT analogs are deprotected using tetrabutylammonium fluoride(TBAF) in tetrahydrofuran. In Step F, the cis and trans hydroxyl PATanalogs are converted in one-pot to the corresponding trans and cisazido PAT intermediates (7) using a Mitsunobu reaction with zincazide/bis-pyridine complex, diisopropyl azodicarboxylate (DIAD) andtriphenylphosphene (Vorogushin et al., 2003). In Step G, the azido PATderivatives are reduced to the corresponding PAT amines (8). In Step H,these enantiomeric cis and trans amines are converted to thedimethylated PAT analogs using Eschweiler-Clarke methylation with formicacid/formaldehyde under reflux. Racemic PATs with Ki<50 nM will beresolved to (+)- and (−)-enantiomers by derivatization of theun-methylated free amine to the diastereomeric salt followed bydifferential crystallization or synthesized de novo using a chiralreduction step. Absolute configuration is assigned by single crystalX-ray crystallography or spectrophotometric methods (NMR, opticalrotation) by comparison to pure enantiomers already synthesized.Products (as HCl salts) characterized for purity using NMR, elementalanalysis, mass spectrometry, melting point and thin layerchromatography.

Example 12

Affinity for 5-HT2c: representative compound results

Ki @ 5HT_(2c) ± PAT # SEM; nM 1 37.6 ± 3.00 2 1270 ± 84.8  3 430 ± 4.8 4   980 ± 7.812 5 ~1800 6 ~1200 7 20.0 ± 1.10 8 ~250 9 ~300 10 ~5,000 13200 14 ~300 15 ~170 16 ~340 17 ~450 24 ~5,000 25 ~5,000

Example 13 Muscarinic Receptor Activity of Compounds of the Invention

Muscarinic receptor activity is assessed using any suitable protocol,including those essentially as described in: Novascreen, NIMHPsychotherapeutic Drug Discovery and Development Program, OceanixBiosciences Corporation, 1996; and PDSP, Psychoactive Drug ScreeningProgram; B L Roth, Director. NIMH Contract NO2MH80002, Case WesternReserve University, Cleveland, Ohio, and University of North Carolina,Chapel Hill, N.C., 2005.

Muscarinic Receptor Activity of PAT Muscarinic Affinity of (+/−)trans-Receptor PAT Ki (nM) M1 2400 M2 M3 1300 M4 M5 600

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The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination of listed elements. The recitation of an element, or anembodiment herein includes that element or embodiment as any singleelement or embodiment or in combination with any other element,embodiments or portions thereof.

All references cited herein, whether in print, electronic, computerreadable storage media or other form, are expressly incorporated byreference in their entirety, including but not limited to, abstracts,articles, journals, publications, texts, treatises, technical datasheets, internet web sites, databases, patents, patent applications, andpatent publications.

Although the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations of theinvention may be devised by others skilled in the art without departingfrom the true spirit and scope of the invention. The claims are intendedto be construed to include all such embodiments and equivalentvariations.

1. A method of treating or preventing a GPCR-mediated disorder in asubject comprising administering to the subject identified as in needthereof a PAT compound.
 2. The method of claim 1, wherein the PATcompound is a compound of Table
 1. 3. The method of claim 1, wherein thePAT compound is:

wherein, R₁ is independently H, NH₂, NH(alkyl), N(alkyl)₂; R₂ isindependently —(CH₂)n-; Each n is independently 1 or 2; R₃ isindependently H, OH, or halo; R₄ is independently H, OH, or halo Each R₅is independently H, alkyl, or halo; R₆ is independently H or alkyl; andR₇ independently H, N(alkyl)₂.
 4. The method of claim 3, wherein in thePAT compound R₁ is —NMe₂.
 5. The method of claim 4, wherein the PATcompound is(1R,3S)-(−)-Trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene.6. The method of claim 1, wherein the disorder is a neuropsychiatricdisorder (e.g., obesity, addiction, anxiety, depression, schizophrenia,and sleep disorders), a neurodegenerative disorder (e.g., Parkinson'sDisease, Alzheimer's Disease), a neurological disorder (e.g., epilepsy),a cardiovascular disorder (e.g., hypertension), a gastrointestinaldisorder (e.g., irritable bowel syndrome), or a genitor-urinary tractdisorder (e.g., bladder control).
 7. The method of claim 1, wherein thedisorder is cocaine addiction.
 8. The method of claim 1, wherein thedisorder is obesity.
 9. A method of inhibiting 5-HT2C in a subjectidentified as in need of such treatment, comprising administering a PATcompound.
 10. A method of treating obesity in a subject comprisingadministering to the subject identified as in need thereof a PATcompound capable of selectively inhibiting the 5-HT2c relative to 5-HT2aor 5-HT2b.
 11. The method of claim 10, wherein the binding interactionthe for inhibiting 5-HT2c is at least 5-fold (alternatively at least10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 500 fold) greater than foreither 5-HT2a or 5-HT2b.
 12. The method of claim 10, wherein the whereinthe binding interaction the for inhibiting 5-HT2c is at least 100-foldgreater than for either 5-HT2a or 5-HT2b.
 13. A method for identifying acompound that is capable of modulating 5-HT2c activity comprising; (i)producing a three-dimensional representation of a molecule or molecularcomplex, wherein said molecule or molecular complex comprises a bindingpocket defined by structure coordinates of 5-HT2c; or b) athree-dimensional representation of a homologue of said molecule ormolecular complex, wherein said homologue comprises a binding pocketthat has a root mean square deviation from the backbone atoms of saidamino acids of not more than about 2.0 angstroms; (ii) producing athree-dimensional representation of a test compound; (iii) assessing thebinding interaction of the test compound with the target.
 14. The methodof claim 13, further comprising contacting the test compound with a5-HT2c and measuring the binding activity of the compound.
 15. Acompound that is:

wherein, R₁ is independently H, NH₂, NH(alkyl), N(alkyl)₂; R₂ isindependently —(CH₂)n-; Each n is independently 1 or 2; R₃ isindependently H, OH, or halo; R₄ is independently H, OH, or halo Each R₅is independently H, alkyl, or halo; R₆ is independently H or alkyl; andR₇ independently H, N(alkyl)₂; or salt, hydrate or solvate thereof. 16.The compound of claim 15, wherein the compound substituents at the1-position and the 3-position are in the trans-orientation to oneanother.
 17. A composition comprising a compound of claim 15 and apharmaceutically acceptable carrier.
 18. A method of making acomposition of claim 17 comprising combining a compound of claim 15 anda pharmaceutically acceptable carrier.
 19. A compound of the formula:

wherein, R₁ is independently H, NH₂, NH(alkyl), N(alkyl)₂; R₂ isindependently —(CH₂)n-; Each n is independently 1 or 2; R₃ isindependently H, OH, or halo; R₄ is independently H, OH, or halo Each R₅is independently H, alkyl, or halo; R₆ is independently H or alkyl; R₇independently H, N(alkyl)₂; and R8 is independently aryl or heteroaryl,each optionally substituted with 1-4 independent R₅; or salt, hydrate orsolvate thereof.